Pancreatic polypeptide family motifs, polypeptides and methods comprising the same

ABSTRACT

The present invention provides novel Pancreatic Polypeptide Family (“PPF”) polypeptides and methods for their use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/640,352, filed Dec. 17, 2009, which is a continuation of U.S.application Ser. No. 11/301,744, filed Dec. 12, 2005, issued as U.S.Pat. No. 7,723,471, which claims the benefit of U.S. Application No.60/635,897 filed Dec. 13, 2004 and is a continuation-in-part of U.S.application Ser. No. 11/055,098, filed Feb. 11, 2005, which claims thebenefit of U.S. Application No. 60/543,406, filed Feb. 11, 2004, andU.S. Application No. 60/543,407, filed Feb. 11, 2004, each of which isherein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

A number of related hormones make up the pancreatic polypeptide family(“PPF”). Pancreatic polypeptide (“PP”) was discovered as a contaminantof insulin extracts and was named by its organ of origin rather thanfunctional importance (Kimmel et al., Endocrinology 83: 1323-30 (1968)).PP is a 36-amino acid peptide (SEQ ID NO: 1) containing distinctivestructural motifs. A related peptide was subsequently discovered inextracts of intestine and named Peptide YY (“PYY”) (SEQ ID NO: 2)because of the N- and C-terminal tyrosines (Tatemoto, Proc. Natl. Acad.Sci. USA 79: 2514-8 (1982)). A third related peptide was later found inextracts of brain and named Neuropeptide Y (“NPY”) (SEQ ID NO: 4)(Tatemoto, Proc. Natl. Acad. Sci. USA 79: 5485-9 (1982); Tatemoto etal., Nature 296: 659-60 (1982)).

These three related peptides base been reported to exert variousbiological effects. Effects of PP include inhibition of pancreaticsecretion and relaxation of the gallbladder. Centrally administered PPproduces modest increases in feeding that may be mediated by receptorslocalized to the hypothalamus and brainstem (reviewed in Gehlert, Proc.Soc. Exp. Biol. Med. 218: 7-22 (1998)).

Release of PYY (SEQ ID NO: 2) occurs following a meal. An alternatemolecular form of PYY is PYY(3-36) (SEQ ID NO: 3) (Eberlein et al.,Peptides 10: 797-803 (1989); Grandt et al., Regul. Pept. 51: 151-9(1994)). This fragment constitutes approximately 40% of total PYY-likeimmunoreactivity in human and canine intestinal extracts and about 36%of total plasma PYY immunoreactivity in a fasting state to slightly over50% following a meal. It is apparently a dipeptidyl peptidase-IV (DPP4)cleavage product of PYY. PYY(3-36) is reportedly a selective ligand atthe Y2 and Y5 receptors, which appear pharmacologically unique inpreferring N-terminally truncated (i.e., C-terminal fragments of) NPYanalogs. Peripheral administration of PYY reportedly reduces gastricacid secretion, gastric motility, exocrine pancreatic secretion(Yoshinaga et al., Am. J. Physiol. 263: G695-701 (1992); Guan et al.,Endocrinology 128: 911-6 (1991); Pappas et al., Gastroenterology 91:1386-9 (1986)), gallbladder contraction and intestinal motility (Savageet al., Gut 28: 166-70 (1987)). The effects of central injection of PYYon gastric emptying, gastric motility and gastric acid secretion, asseen after direct injection in or around the hindbrain/brainstem (Chenand Rogers, Am. J. Physiol. 269: R787-92 (1995); Chen et al., Regul.Pept. 61: 95-98 (1996); Yang and Tache, Am. J. Physiol. 268: G943-8(1995); Chen et al., Neurogastroenterol. Motil. 9: 109-16 (1997)), maydiffer from those effects observed after peripheral injection. Forexample, centrally administered PYY had some effects opposite to thosedescribed herein for peripherally injected PYY(3-36) in that gastricacid secretion was stimulated, not inhibited. Gastric motility wassuppressed only in conjunction with TRH stimulation, but not whenadministered alone, and was indeed stimulatory at higher doses throughpresumed interaction with PP receptors. PYY has been shown to stimulatefood and water intake after central administration (Morley et al., BrainRes. 341: 200-3 (1985); Corp et al., Am. J. Physiol. 259; R317-23(1990)).

Likewise, one of the earliest reported central effects of NPY (SEQ IDNO: 4) was to increase food intake, particularly in the hypothalamus(Stanley et al., Peptides 6: 1205-11 (1985)). PYY and PP are reported tomimic these effects, and PYY is more potent or as potent as NPY (Morleyet al., Brain Res. 341: 200-3 (1985); Kanatani et al., Endocrinology141: 1011-6 (2000); Nakajima et al., J. Pharmacol. Exp. Ther. 268:1010-4 (1994)). Several groups found the magnitude of NPY-inducedfeeding to be higher than that induced by any pharmacological agentpreviously tested, and also extremely long-lasting. NPY-inducedstimulation of feeding has been reproduced in a number of species. Amongthe three basic macronutrients (fat, protein, and carbohydrate), theintake of carbohydrates was preferentially stimulated. No tolerance wasseen towards the orexigenic effect of NPY, and when administration ofthe peptide was repeated over 10 days, a marked increase in the rate ofweight gain was observed. Following starvation, the concentration of NPYin the hypothalamic PVN increased with time, and returned rapidly tocontrol levels following food ingestion.

Pharmacological studies and cloning efforts have revealed a number ofseven transmembrane receptors for the PP family of peptides, and thesereceptors have been assigned the names Y1 through Y6 (and a putativePYY-preferring receptor Y7). Typical signaling responses of thesereceptors are similar to those of other G_(i)/G₀-coupled receptors,namely inhibition of adenylate cyclase. Even with fairly low sequencehomology among receptors, it is apparent that there is a clustering ofamino acid sequence similarity between Y1, Y4 and Y6 receptors, while Y2and Y5 define other families. Other binding sites have been identifiedby the rank order of potency of various peptides. The NPY-preferringreceptor, which has not been cloned, has been termed Y3, and there isevidence for the existence of PYY-preferring receptors (the putative Y7receptor(s)) (reviewed in Michel et al., Pharmacol. Rev. 50:141-50(1998); Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22 (1998)).

The Y5 and Y1 receptors have been suggested as the primary mediators ofthe food intake response (Marsh et al., Nat. Med. 4: 718-21 (1998);Kanatoni et al., Endocrinology 141: 1011-6 (2000)). The prevalent ideahas been that endogenous NPV, via these receptors, increases feedingbehavior. Proposed therapies for obesity have invariably been directedtoward antagonism of NPY receptors, while therapies for treatinganorexia have been directed toward agonists of this ligand family (see,e.g., U.S. Pat. Nos. 5,939,462; 6,013,622; and 4,891,357). In general,PYY and NPY are reported to be equipotent and equally effective in allY1, Y5 (and Y2) receptor assays studied (Gehlert, Proc. Soc. Exp. Biol.Med. 218: 7-22 (1998)).

Pharmacologically, the Y2 receptor is distinguished from Y1 byexhibiting affinity for C-terminal fragments of neuropeptide Y. The Y2receptor is most often differentiated by the affinity of neuropeptideY(13-36), although the 3-36 fragment of neuropeptide Y and peptide YYprovided improved affinity and selectivity (see Dumont et al., Soc. forNeurosci. Abstracts 19:726 (1993)). Signal transmission through both theY1 and Y2 receptors are coupled to the inhibition of adenylate cyclase.Binding to the Y2 receptor was also found to reduce the intracellularlevels of calcium in the synapse by selective inhibition of N-typecalcium channels. In addition, the Y2 receptor, like the Y1 receptors,exhibits differential coupling to second messengers (see U.S. Pat. No.6,355,478). Y2 receptors are found in a variety of brain regions,including the hippocampus, substantia nigra-lateralis, thalamus,hypothalamus, and brainstem. The human, murine, monkey and rat Y2receptors have been cloned (e.g., see U.S. Pat. No. 6,420,532 and U.S.Pat. No. 6,355,478).

The main characteristic of putative Y3 receptors is that they recognizeNPY, while PYY is at least an order of magnitude less potent. The Y3receptor represents the only binding site/receptor that shows apreference for NPY.

There is an additional binding site/receptor which shows preference forPYYs, termed PYY-preferring receptor, which is referred to herein as theY7 receptor(s). Different rank orders of binding to this receptor, orclass of receptors, have been reported, suggesting that there may bemore than one receptor in this class. In most cases it has been appliedto describe a receptor where PYY was three to live times more potentthan NPY. The International Union of Pharmacology recommendations forthe nomenclature of NPY, PYY and PP receptors are that the termPYY-preferring receptor is not used unless a potency difference of atleast twenty-fold between PYY and NPY is observed (Michel et al.,Pharmacol. Rev. 50: 143-50 (1998)). However, for purposes of thisdisclosure, reference to the Y7 receptor or pharmacology of aPYY-preferring receptor means a receptor having any degree of preferencefor PYY over NPY.

Obesity and its associated disorders are common and very serious publichealth problems in the United States and throughout the world. It isestimated that about 64% of Americans are overweight or obese (roughlyabout 97 million adults) and it is generally believed that these numbersare increasing. People who are overweight or obese are considered thosewith a Body Mass Index (BMI) equal to or greater than 25. BMI is amathematical formula commonly used to express the relationship ofweight-to-height; a person's body weight in kilograms is divided by thesquare of his or her height in meters (i.e., wt/(ht)²). In a humanhealthcare setting, individuals with a BMI of 25 to 29.9 are generallyconsidered overweight, while individuals with a BMI of 30 or more aregenerally considered obese. Morbid obesity refers to a BMI of 40 orgreater. According to the NIH Clinical Guidelines on the Identification,Evaluation, and Treatment of Overweight and Obesity in Adults, alladults (aged 18 years or older) who have a BMI of 25 or more areconsidered at risk for premature death and disability as a consequenceof overweight and obesity. These health risks increase even more as theseverity of an individual's obesity increases.

Being obese or overweight may substantially increase the risk ofmorbidity from hypertension; dyslipidemia; type 2 diabetes; coronaryheart disease; stroke; gallbladder disease; osteoarthritis; sleep apneaand respiratory problems; and endometrial, breast, prostate, and coloncancers. Higher body weights are also associated with increases inall-cause mortality. Furthermore, being obese or overweight may cause aperson to have a negative self-image about him or her self.

Upper body obesity is the strongest risk factor known for type 2diabetes mellitus, and is a strong risk factor for cardiovasculardisease. Obesity is a recognized risk factor for hypertension,atherosclerosis, congestive heart failure, stroke, gallbladder disease,osteoarthritis, sleep apnea, reproductive disorders such as polycysticovarian syndrome, cancers of the breast, prostate, and colon, andincreased incidence of complications of general anesthesia (see, e.g.,Kopeleman, Nature 404; 635-43 (2000)). It reduces life-span and carriesa serious risk of co-morbidities above, as well as disorders such asinfections, varicose veins, acanthosis nigricans, eczema, exerciseintolerance, insulin resistance, hypertension hypercholesterolemia,cholelithiasis, orthopedic injury, and thromboembolic disease (Rissanenet al., Br. Med. J. 301: 835-7 (1990)). Obesity is also a risk factorfor the group of conditions called insulin resistance syndrome, or“Syndrome X.” Recent estimate for the medical cost of obesity andassociated disorders is $150 billion worldwide. The pathogenesis ofobesity is believed to be multifactoral; generally, in obese oroverweight subjects, when nutrient availability and energy expenditureequilibrate, an excess of adipose tissue results. Obesity is currently apoorly treatable, chronic, essentially intractable metabolic disorder. Atherapeutic drug useful in weight reduction of obese persons could havea profound beneficial effect on their health.

For these reasons, there is an enormous interest in treating obesity.Existing therapies include standard diets and exercise, very low caloriediets, behavioral therapy, pharmacotherapy involving appetitesuppressants, thermogenic drugs, food absorption inhibitors, mechanicaldevices such as jaw wiring, waist cords and balloons, and surgery, suchas gastric bypass, Jung and Chong, Clinical Endocinology, 35:11-20(1991); Bray, Am. J. Clin. Nutr. 55:538S-544S (1992).

In addition to the interest in treating obesity for physical health, thedrive to look good and feel good about oneself has always been ofinterest and is a lucrative market. It has been reported by the AmericanSociety for Aesthetic Plastic Surgery that 6.9 million cosmeticprocedures were performed in 2002. Liposuction was the most commonsurgical procedure. Moreover, the National Center for Health Statisticsreported that, in 2002, about a third of all adult Americans engaged inregular leisure-time physical activity.

In general, while loss of fat is desired, loss of lean body mass(protein) is not. Lean body mass is highly active metabolically andphysiologically. Lean body mass contains all the body protein. There isno real protein store as every protein molecule has a role inmaintaining homeostasis. It is believed that loss of body protein isdeleterious to the health of on individual. The majority of protein inthe lean body mass is in the skeletal muscle mass. Lean body mass is50-60% muscle mass by weight, the rest is bone and tendon. Protein makesup the critical cell structure in muscle, viscera, red cells andconnective tissue. Enzymes, which direct metabolism, and antibodies,which maintain immune function, are also proteins. Moreover, a body withgreater lean body mass to fat ratio may be more aesthetically pleasingto some individuals. Thus, it is desirable to prevent or minimize lossof lean body mass, even while reducing body fat.

Caloric restriction, regardless of its form, can cause catabolism ofbody protein and produce negative nitrogen balance. Protein-supplementeddiets, therefore, have gained popularity as a means of lesseningnitrogen loss during caloric restriction. Protein-sparing modifiedfasting has been reported to be effective in weight reduction inadolescents. Lee et al. Clin. Pediatr. 31:234-236 (April 1992). However,these diets may produce only modest nitrogen sparing.

There remains a need to develop further PYY analog polypeptides.Accordingly, it is an object of the present invention to provide suchPYY analog polypeptides and methods for producing and using them. A needexists for effective ways of promoting fat loss yet preserving lean bodymass or minimizing its loss. Described herein are novel methods formodifying body composition.

All documents referred to herein are incorporated by reference into thepresent application as though fully set forth herein.

SUMMARY OF THE INVENTION

The present invention relates generally to pancreatic polypeptide(“PPF”) polypeptides having at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 92%, at least 94% or at least 97%sequence identity to PYY(3-36) over the entire length of PYY(3-36), andalso comprise at least two PPF motifs including at least the N-terminalpolyproline PPF motif and the C-terminal tail PPF motif. Additional PPFmotifs of the invention may correspond to any motif of any of the PPfamily polypeptides, including PP, PYY and NPY. In certain embodiments,the PPF polypeptides do not include unnatural amino acids. In otherembodiments, the PPF polypeptides do not include known naturallyoccurring species variants.

In one aspect, the PPF polypeptides of the invention include PYY analogpolypeptides. In yet another aspect of the invention, the PPFpolypeptides of the invention include PPF chimeric polypeptidescomprising a fragment of a PP, PYY or NPY polypeptide covalently linkedto at least one additional fragment of a PP, PYY or NPY polypeptide,wherein each PP, PYY or NPY fragment includes a PPF motif. Such PPFanalog polypeptides and PPF chimeric polypeptides of the invention willexhibit at least 50% sequence identity to a native PYY(3-36) over theentire length of the PYY(3-36). In certain embodiments, desirable PPFchimeric polypeptides include an N-terminal PP fragment in combinationwith a C-terminal PYY fragment. In other embodiments, PPF chimericpolypeptides include an N-terminal PP fragment in combination with aC-terminal NPY fragment. In other embodiments, PPF chimeric polypeptidesinclude an N-terminal PYY fragment and a C-terminal PP or NPY fragment.In other embodiments, PPF chimeric polypeptides include an N-terminalNPY in combination with a C-terminal PYY or PP. In other embodiments,PPF chimeric polypeptides may not include an N-terminal PP fragment incombination with a C-terminal NPY fragment. In still other embodiments,PPF chimeric polypeptides may not include an N-terminal NPY fragmentwith a C-terminal PYY fragment.

In another aspect of the invention, methods for treating or preventingobesity are provided, wherein the method comprises administering atherapeutically or prophylactically effective amount of a PPFpolypeptide of the invention to a subject in need thereof. In someembodiments, the subject is an obese or overweight subject. While“obesity” is generally defined as a body mass index over 30, forpurposes of this disclosure, any subject, including those with a bodymass index of less than 30, who needs or wishes to reduce body weight isincluded in the scope of “obese.” Subjects who are insulin resistant,glucose intolerant, or have any form of diabetes mellitus (e.g., type 1,2 or gestational diabetes) can benefit from this method.

In one general aspect, methods of the invention include the use of a PPFpolypeptide to modify body composition, for example, reducing body fat,but not lean body mass. The change in body composition can be by, forexample, weight (e.g., loss or gain by grams), by percent body fat andpercent lean body mass or protein (used interchangeably), or by theratio of body fat to lean tissue.

While it has been reported that PYY may be useful in regulating satiety(U.S. Pat. No. 6,538,708) or control of weight (U.S. patent applicationSer. No. 10/016,969, WO2003026591 and WO2003057235), it has nowsurprisingly been discovered that PPF polypeptides may have a metaboliceffect on the body and may be used to affect body composition, leadingto the desirable loss of body fat, yet preserving lean body mass orminimizing its loss.

In certain embodiments, methods of the invention include reducing bodyfat or reducing or preventing body fat gain, while sparing, minimizingloss, or even increasing lean body mass. Other embodiments includecontrolling body weight and/or sculpting a body's appearance. Thesubjects to whom these methods may be of interest are those individualswho are overweight or obese, as well as those who are lean. Forinstance, subjects with lean body composition, e.g., body builders andother athletes, may benefit from the invention as well. It may bedesirable for them to reduce or maintain their body weight, e.g., tostay in a certain weight class range, yet preserve or increase theirlean body mass for greater strength, stamina, endurance and/or a moremuscular appearance. Such methods may also be used on any animal forwhich a greater lean body mass to fat ratio is desired. Examples of suchuse include, but are not limited to, creating a superior show dog orcreating a superior racehorse or workhorse.

In one general aspect, methods of the invention include the use of a PPFpolypeptide to reduce the fat content in animals for consumption.Methods of the invention can include producing a leaner meat source.Compositions and methods of the invention can be used with livestockincluding, but not limited to, chicken, turkeys, cows, pigs, and sheep.

It is contemplated that methods of the invention can be used incombination with other forms of nutritional regimens and weight lossprograms, such as those already described above, for example, those thatinclude life-style changes that include monitoring food intake (quantityand quality) and exercising, as well as surgery. Nutritional regimensinclude those that are used to increase lean body mass such as thosefollowed by body builders.

In another general aspect, PPF polypeptides reduce the respiratoryquotient (RQ) in animals, which is indicative of improved fatutilization for energy at the tissue and cell level (increased fattyacid β-oxidation). Thus, PPF polypeptides may be therapeutically usefulin conditions where improved fatty acid β-oxidation in non-adiposetissues is desirable with maintenance, minimization of loss, or anincrease in lean body mass. Examples of such conditions include, but arenot limited to, nonalcoholic steatohepatitis (NASH) (Grant et al.Nonalcoholic fatty liver disease. Ann Hepatol. 3(3):93-9 July-September2004), in which patients display pathologically elevated liver fatcontent, and lipodystrophy, in which patients lack significant adiposestores, and hence display increased fat build-up in non-adipose tissuessuch as liver and skeletal muscle (Garg et al. Lipodystrophies: raredisorders causing metabolic syndrome, Endocrinol Metab Clin North Am.33(2):305-31 June 2004).

In certain embodiments of the invention, a PPF polypeptide may beadministered peripherally and not centrally, i.e, not through thecentral nervous system. In other embodiments, a therapeutically orprophylactically effective amount of a PPF polypeptide is administeredin a single dose, multiple doses, or continuous administration.

In yet another aspect of the invention, compounds of the invention canbe used for methods of reducing food intake, reducing nutrientavailability, causing weight loss, affecting body composition, alteringbody energy content or energy expenditure (EE) and improving lipidprofile (including reducing LDL cholesterol and/or triglyceride levelsand/or changing HDL cholesterol levels). Thus, in certain embodiments,the methods of the invention are useful for treating or preventingconditions or disorders which can be alleviated by reducing nutrientavailability in a subject in need thereof, comprising administering tosaid subject a therapeutically or prophylactically effective amount of aPPF polypeptide of the invention. Such conditions and disorders include,but are not limited to, hypertension, dyslipidemia, cardiovasculardisease, eating disorders, insulin-resistance, obesity, diabetesmellitus of any kind, including Type I, Type II, and gestationaldiabetes. Compounds of the invention may also be useful in treating orpreventing other conditions associated with obesity including stroke,cancer (e.g., endometrial, breast, prostate, and colon cancer),gallbladder disease, sleep apnea, reduced fertility, and osteoarthritis,(see Lyznieki et al. Am. Fam. Phys. 63:2185, 2001).

Compounds of the invention may also be useful for potentiating,inducing, enhancing or restoring glucose responsivity in pancreaticislets or cells. These actions may also be used to treat or preventconditions associated with metabolic disorders such as those describedabove and in U.S. patent application no. US20040228846.

In addition to the amelioration of hypertension in subjects in needthereof, compounds of the invention may be used to treat or preventhypotension.

Compounds of the invention may also be useful in the treatment orprevention of any number of gastrointestinal disorders that areassociated with excess intestinal electrolytes and water secretion aswell as decreased absorption, e.g., infectious (e.g., viral orbacterial) diarrhea, inflammatory diarrhea, short bowel syndrome, or thediarrhea which typically occurs following surgical procedure e.g.,ileostomy (see e.g., Harrison's principles of Internal Medicine, McGrawHill Inc., New York, 12th ed.). Examples of infectious diarrhea include,without limitation, acute viral diarrhea, acute bacterial diarrhea(e.g., salmonella, campylobacter, and clostridium) or diarrhea due toprotozoal infections, or travelers' diarrhea (e.g., Norwalk virus orrotavirus). Examples of inflammatory diarrhea include, withoutlimitation, malabsorption syndrome, tropical spue, chronic pancreatitis,Crohn's disease, diarrhea, and irritable bowel syndrome. It has alsobeen discovered that the peptides of the invention can be used to treator prevent an emergency or life-threatening situation involving agastrointestinal disorder, e.g., after surgery or due to cholera.Furthermore, the compounds of the invention can be used to treatintestinal dysfunction in patients with Acquired Immune DeficiencySyndrome (AIDS), especially during cachexia. The compounds of theinvention may also be useful for inhibiting small intestinal fluid andelectrolyte secretion, and augmenting nutrient transport, as well asincreasing cell proliferation in the gastrointestinal tract, regulatinglipolysis in, e.g., adipose tissue and regulating blood flow in amammal.

Compounds of the invention may also be useful for treating or preventingthe above conditions by their gastrointestinal protective activity.Accordingly, compounds of the invention may be used to treatgastrointestinal or mucosal damage. Exemplary types of damage include,but are not limited to, inflammatory bowel disease, bowel atrophy,conditions characterized by loss of bowel mucosa or bowel mucosalfunction, and other conditions of the gastrointestinal tract, includingthose which may be brought about by exposure to cytotoxic agents,radiation, toxicity, infection and/or injury. Moreover, these compoundsof the invention may be combined with analgesics, anti-inflammatoryagents, growth hormone, heparin, or any other therapies that may be usedto treat inflammatory bowel disease or other conditions listed above.

Moreover, compounds of the invention are useful in treating orpreventing diseases and disorders that can be alleviated or amelioratedby their anti-secretory properties. Such anti-secretory propertiesinclude inhibition of gastric and/or pancreatic secretions and can beuseful in the treatment or prevention of diseases and disordersincluding gastritis, pancreatitis. Barrett's esophagus, andGastroesophageal Reflux Disease. These diseases may also be treated orprevented by the gastrointestinal protective functions of compounds ofthe invention.

Compounds of the invention may also be useful for reducing aluminumconcentrations in the central nervous system of a subject to treat orprevent a disease or condition associated with abnormal aluminumconcentrations (e.g., a patient afflicted with Alzheimer's disease or atrisk for developing Alzheimer's disease, dialysis dementia, or increasedaluminum levels due to occupational exposure).

The present invention also relates to pharmaceutical compositionscomprising a therapeutically or prophylactically effective amount of atleast one PPF polypeptide of the invention, or a pharmaceuticallyacceptable salt thereof, together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers useful in the delivery of the PPF polypeptides.

These and other aspects of the invention will be more clearly understoodwith reference to the following embodiments and detailed description.The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. All references citedherein are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B demonstrate the activity of certain PPF polypeptides ofthe invention in a food intake assay.

FIGS. 2A and 2B demonstrate the activity of additional PPF polypeptidesof the invention in a food intake assay.

FIGS. 3A and 3B demonstrate the activity of yet additional PPFpolypeptides of the invention in a food intake assays.

FIG. 4 demonstrates the activity of yet additional PPF polypeptides ofthe invention in a food intake assay.

FIGS. 5A and 5B demonstrate the activity of certain PPF polypeptides ofthe invention in the diet-induced obese (DIO) mouse model.

FIG. 6 demonstrates the activity of additional PPF polypeptides of theinvention in the DIO mouse model.

FIG. 7 shows weight gain in rats.

FIGS. 8A and 8B demonstrate the activity of a PPF polypeptide of theinvention in a food intake assay in the DIO mouse model, as compared toPYY(3-36).

FIGS. 9A-9D demonstrate the effect of PPF polypeptides of the inventionon heart rate and blood pressure, as compared to PYY and PYY(3-36).

FIG. 10 demonstrates the activity of PPF polypeptides of the inventionon gastric acid secretion.

FIG. 11 demonstrates the activity of PPF polypeptides of the inventionon gastric acid secretion.

FIGS. 12-17 demonstrate the activity of PPF polypeptides of theinvention on gastric emptying.

FIG. 18 demonstrates the activity of PPF polypeptides of the inventionon gallbladder emptying.

FIG. 19 demonstrates the activity of PPF polypeptides of the inventionon gallbladder emptying.

FIG. 20 demonstrates the activity of PPF polypeptides of the inventionon gastric mucosal protection.

FIGS. 21A and 21B depict an exemplary effect of PYY(3-36) administrationon body weight in DIO mice.

FIGS. 22A and 22B depict an exemplary effect of PYY(3-36) administrationon food intake in the mice of FIGS. 21A and 21B, respectively.

FIGS. 23A and 23B depict an exemplary effect of PYY(3-36) administrationon respiratory quotient (RQ) during light and dark cycles in the mice ofFIG. 21A.

FIGS. 24A and 24B depict an exemplary effect of PYY(3-36) administrationon epididymal fat pad weight in the mice of FIGS. 21A and 21B,respectively.

FIGS. 25A and 25B depict an exemplary effect of PYY(3-36) administrationon fat and lean tissue mass in the mice of FIG. 21B.

FIG. 26 depicts exemplary effects of PYY(3-36) administration on bodyweight at various doses in DIO mice versus high-fat fed and low-fat fedcontrol mice.

FIG. 27 depicts an exemplary effect of PYY(3-36) administration onweekly food intake in the mice of FIG. 26.

FIGS. 28A and 28B depict exemplary effects of PYY(3-36) administrationon fat and lean tissue mass in the mice of FIG. 26.

FIGS. 29A and 29B depict exemplary effects of PYY(3-36) administrationon body weight and food intake in DIO mice.

FIG. 30 depicts an exemplary effect of PYY(3-36) administration onepididymal fat pad weight in the mice of FIGS. 29A and 29B.

FIGS. 31A and 31B depict exemplary effects of PYY(3-36) administrationon fat and lean tissue mass in the mice of FIGS. 29A and 29B.

FIGS. 32A and 32B depict exemplary effects of PYY(3-36) administrationon metabolic rate during light and dark cycles in DIO mice.

FIG. 33 depicts exemplary effects of various PYY(3-36) concentrations ongallbladder weight in non-obese mice.

FIGS. 34A and 34B depict exemplary effects of prolonged PYY(3-36)administration and withdrawal in DIO mice.

FIGS. 35A and 35B depict PYY(3-36) dose responsive decreases in foodintake and body weight in DIO prone rats.

FIGS. 36A-D depict exemplary effects of PYY(3-36) with and withoutco-administration of amylin on fasting plasma parameters in DIO pronerats.

FIGS. 37A-C depict exemplary effects of PYY(3-36) with and withoutco-administration of amylin on respiratory quotient (RQ) and energyexpenditure (EE) in DIO prone rats.

FIGS. 38A-C depict exemplary effects of PYY(3-36) with and withoutco-administration of amylin on body composition in DIO prone rats.

FIG. 39 compares the calculated rate of degradation of an exemplary PPFpolypeptide to that of PYY(3-36).

FIGS. 40A and 40B demonstrate exemplary effects of acute administrationof a PPF polypeptide in food intake assays in mouse and rat models, ascompared to PYY(3-36).

FIGS. 41A and 41B demonstrate exemplary effects of chronicadministration of a PPF polypeptide on body weight in rodent DIO models,as compared to PYY(3-36).

FIGS. 42A-D depict effects of administration of an exemplary PPFpolypeptide on feeding pattern in a rat model.

FIG. 43 depicts effects of administration of an exemplary PPFpolypeptide on body composition, as compared to PYY(3-36), in DIO rats.

FIG. 44 depicts effects of administration of an exemplary PPFpolypeptide on triglyceride levels, as compared to PYY(3-36), in DIOrats.

FIGS. 45A and 45B depict effects of administration of an exemplary PPFpolypeptide on gastric emptying, as compared to PYY(3-36), in rats.

FIGS. 46A and 46B depict effects of administration of an exemplary PPFpolypeptide on heart rate and mean arterial pressure (MAP) in rats.

FIGS. 47A and 47B depict effects of administration of an exemplary PPFpolypeptide on heart rate and mean arterial pressure (MAP) in rats.

FIGS. 48A and 48B depict effects of an exemplary PPF polypeptide, ascompared to PYY(3-36) with and without co-administration of amylin onbody weight in DIO prone rats.

FIGS. 49A and 49B depict effects of two exemplary PPF polypeptides withand without co-administration of amylin on body weight in DIO pronerats.

FIGS. 50A-C depict effects of an exemplary PPF polypeptide with andwithout co-administration of amylin on body composition in DIO pronerats.

FIGS. 51A-C depict effects of an exemplary PPF polypeptide with andwithout co-administration of amylin on body composition in DIO pronerats.

FIGS. 52A and 52B depict effects of PYY(3-36) or an exemplary PPFpolypeptide with and without co-administration of amylin on fastinginsulin levels in rats.

FIGS. 53A and 53B depict effects of an exemplary PPF polypeptide withand without co-administration of amylin on RQ and EE in rats.

FIG. 54 compares the calculated rates of degradation of several PPFpolypeptides to that of PYY(3-36).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to pancreatic polypeptide family(“PPF”) polypeptides having at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 92%, at least 94% or at least 97%sequence identity to PYY(3-36) over the entire length of PYY(3-36). ThePPF polypeptides may comprise not more than 10, not more than 5, notmore than 3, not more than 2 or not more than 1 amino acidsubstitutions. The PPF polypeptides also comprise at least two PPFmotifs including at least the N-terminal polyproline PPF motif and theC-terminal tail PPF motif. As used herein, “motif” refers to an aminoacid sequence that is characteristic of a specific biochemical functionor defines an independently folded domain. Additional PPF motifs of theinvention may correspond to a motif of any of the PP familypolypeptides, including PP, PYY and NPY, for example the type II β-turnregion motif of PYY, or the α-helical motif at the C-terminal end ofPYY. In certain embodiments, the PPF polypeptides of the invention maynot include any unnatural amino acids.

The present invention also relates to PPF polypeptides useful in thetreatment and prevention of metabolic conditions and disorders. In someembodiments, the PPF polypeptides of the invention may have comparableor higher potency in the treatment and/or prevention of metabolicconditions and disorders, as compared to native human PP, PYY, PYY(3-36)or NPY. In some embodiments, PPF polypeptides of the invention mayexhibit less potency but may possess other desirable features such asimproved ease of manufacture, stability, and/or ease of formulation, ascompared to PP, PYY, PYY(3-36), or NPY.

In some embodiments, and without intending to be limited by theory, itis believed that the peripheral administration of the novel PPFpolypeptides of the invention to a subject reduces nutrientavailability, and thus is useful in the treatment and prevention ofobesity and related metabolic conditions or disorders. As such, thepresent invention provides PPF polypeptide compositions and methods ofusing them to reduce nutrient availability in a subject in need thereoffor treating and preventing metabolic conditions or disorders that maybenefit from a reduction in nutrient availability. These methods may beuseful in the treatment of, for example, obesity, diabetes, includingbut not limited to type 2 or non-insulin dependent diabetes, eatingdisorders, insulin-resistance syndrome, cardiovascular disease, or acombination of such conditions.

It has now been discovered that a PYY, PYY agonist or PPF polypeptidemay have metabolic effects on the body and may be used to preferentiallyreduce or maintain body fat and spare or increase lean body mass.

The present invention is directed, in part, to affecting bodycomposition by reducing body weight, maintaining body weight, orreducing body weight gain, while selectively reducing body fat orreducing or preventing body fat gain and maintaining or increasing leanbody mass. In certain situations, however, e.g., body building, it maybe desirable to increase body weight, for example, through selectivenutrient intake (e.g., increasing the caloric or fat content), whilereducing or maintaining percent body fat.

The methods of the invention contemplate the administration of aneffective amount of a PYY, PYY agonist or PPF polypeptide to a subjectto affect the desired results as described in the present application.

The administered PYY, PYY agonist or PPF polypeptide may be in the formof a peptide, a prodrug, or as pharmaceutical salts thereof. The term“prodrug” refers to a compound that is a drug precursor that, followingadministration, releases the drug in vivo via some chemical orphysiological process, for example, proteolytic cleavage, or uponreaching an environment of a certain pH.

Methods of the invention can be used on any individual in need of suchmethods or individuals for whom practice of the methods is desired.These individuals may be any mammal including, but not limited to,humans, dogs, horses, cows, pigs, chickens, turkeys and othercommercially valuable or companion animals.

The section headings are used herein for organizational purposes only,and are not to be construed as in any way limiting the subject matterdescribed.

PPF Polypeptides of the Invention and PPF Motifs

As discussed above, the present invention relates, at least in part, tonovel PPF polypeptides comprising at least two PPF motifs, wherein theat least two PPF motifs include at least the N-terminal polyproline PPFmotif and the C-terminal tail PPF motif. The PPF polypeptides of theinvention will also exhibit at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 92%, at least 94% or at least 97%sequence identity to a native PYY(3-36) over the entire length of thePYY(3-36). In some embodiments, the polypeptides of the presentinvention will retain, at least in part, a biological activity of nativehuman PP, PYY or NPY, e.g., the polypeptides of the present inventionwill generally be PP, PYY and/or NPY agonists or antagonists. In someembodiments, the polypeptides of the present invention will exhibitbiological activity in the treatment and prevention of metabolicconditions and disorders. Further, the PPF polypeptides of the inventionmay include internal linker compounds, may include chemicalmodifications at internal amino acid residues, or may be chemicallymodified at the N-terminal or C-terminal residue. In some embodiments,the polypeptides of the invention include only natural L amino acidresidues and/or modified natural L amino acid residues. In someembodiments, the polypeptides of the invention do not include unnaturalamino acid residues.

The PPF motifs of the invention may correspond to any motif of any ofthe native PP family polypeptides, including PP, PYY and NPY. A “PPFmotif” is generally a structural component, primary, secondary, ortertiary, of a native PP family polypeptide that is critical tobiological activity, i.e., biological activity is substantiallydecreased in the absence or disturbance of the motif. PPF motifs caninclude any of those known in the art, including, but not limited to,the N-terminal polyproline type II motif of a native PP familypolypeptide, the type II β-turn motif of native PP family polypeptide,the α-helical motif at the C-terminal end of native PP familypolypeptide, and the C-terminal tail motif of native PP familypolypeptide. More particularly, in the N-terminal polyproline PPF motif,amino acids corresponding to residues 5 and 8 of a native PP familypolypeptide are generally conserved as a proline. The type II β-turnmotif will generally include amino acids corresponding to residues 12-14of a native PP family polypeptide. The α-helical motif can generallyextend from amino acids corresponding to approximately residue 14 of anative PP family polypeptide to any point up to and including theC-terminal end, so long as the α-helical motif includes a sufficientnumber of amino acid residues such that an α-helical turn is formed insolution. The α-helical motif can also include amino acid substitutions,insertions and deletions to the native PP family sequence, so long asthe α-helical turn is still formed in solution. The C-terminal tail PPFmotif generally includes amino acids corresponding to approximately thelast 10 residues of a native PP family polypeptide. In some embodiments,the C-terminal tail motif includes the last 7, 6, or 5 residues of anative PP family polypeptide. In some embodiments, the C-terminal tailmotif includes amino acid residues 32-35.

In one embodiment, the PPF polypeptides of the invention do not includeany unnatural amino acid resides, and further with the provisio that thePPF polypeptides of the invention do not include any native PPFpolypeptides (e.g., PP, NPY(1-36), NPY(3-36), PYY(1-36), PYY(3-36),NPY(2-36), NPY(4-36), PYY(2-36), PYY(4-36), PP(2-36), PP(3-36), orPP(4-36)). In some embodiments, the PPF polypeptides of the invention donot include: Tyr¹hPP, Lys⁴hPP, Asn⁷hPP, Arg¹⁹hPP, Tyr²¹hPP, Glu²¹hPP,Ala²³hPP, Gln²³hPP, Gln³⁴hPP, Phe⁶Arg¹⁹hPP, Phe⁶Tyr²¹hPP, Phe⁶Glu²¹hPP,Phe⁶Ala²³hPP, Phe⁶Gln²³hPP, Pro¹³Ala¹⁴hPP, Ile³¹Gln³⁴PP,Arg¹⁹Tyr²⁰Tyr²¹Ser²²Ala²³hPP, Lys⁴Arg¹⁹Tyr²⁰Tyr²¹Ser²²Ala²³hPP,Lys⁴Arg¹⁹Tyr²⁰Tyr²¹Ser²²Ala²³hPP(2-36), Ala¹NPY, Tyr¹NPY, Ala²NPY,Leu²NPY, Phe²NPY, His²NPY, Ala³NPY, Ala⁴NPY, Ala⁶NPY, Tyr⁷NPY, Ala⁷NPY,Ala⁹NPY, Ala¹⁰NPY, Ala¹¹NPY, Gly¹²NPY, Ala¹³NPY, Gly¹⁴NPY, Ala¹⁵NPY,Ala¹⁶NPY, Ala¹⁷NPY, Gly¹⁸NPY, Ala¹⁹NPY, Lys¹⁹NPY, Ala²⁰NPY, Ala²¹NPY,Ala²²NPY, Gly²³NPY, Ala²⁴NPY, Trp²⁴pNPY, Ala²⁵NPY, Lys²⁵NPY, Ala²⁶NPY,Ala²⁷NPY, Phe²⁷NPY, Ala²⁸NPY, Ala²⁹NPY, Gln²⁹NPY, Ala³⁰NPY, Phe³⁰NPY,Ala³¹NPY, Trp³¹pNPY, Ala³²NPY, Trp³²NPY, Ala³³NPY, Lys³³NPY, Ala³⁴NPY,Pro³⁴NPY, Leu³⁴NPY, Ala³⁵NPY, Lys³⁵NPY, Ala³⁶NPY, Phe³⁶NPY, His³⁶NPY,Glu⁴Pro³⁴pNPY, Arg⁶Pro³⁴pNPY, Phe⁶Pro³⁴pNPY, Cys⁶Pro³⁴pNPY,Asn⁶Pro³⁴pNPY, Phe⁷Pro³⁴pNPY, Arg⁷Pro³⁴pNPY, Cys⁷Pro³⁴pNPY,Asp⁷Pro³⁴pNPY, Phe⁸Pro³⁴pNPY, Arg⁸Pro³⁴pNPY, Cys⁸Pro³⁴pNPY,Asp⁸Pro³⁴pNPY, Asp⁸Pro³⁴pNPY, Pro¹¹Pro³⁴pNPY, Ser¹³Pro¹⁴pNPY,Trp^(24,31)pNPY, Ala³¹Pro³²pNPY, Cys³¹Pro³⁴pNPY, Leu³¹Pro³⁴NPY,Phe³²Pro³⁴pNPY, Ala^(21,25)Pro³⁴pNPY, Pro¹¹Tyr¹³Pro¹⁴Pro³⁴pNPY,Ahx(9-22)pNPY, Ahx(9-17)pNPY, des-AA(10-20)-Cys^(7,21)Pro³⁴-pNPY,des-AA(10-17)-pNPY, des-AA(10-17)-Cys^(2,27)-pNPY,des-AA(10-17)-Ala^(7,21)-pNPY, des-AA(10-17)-Cys^(7,21)-pNPY,des-AA(10-17)-Glu⁷Lys²¹-pNPY, des-AA(10-17)Cys^(7,21)Pro³⁴pNPY,des-AA(10-17)Glu⁷Lys²¹Pro³⁴pNPY, des-AA(10-17)Cys^(7,21)Leu³³Pro³⁴pNPY,des-AA(11-17)Cys^(7,21)Pro³⁴pNPY, Pro³⁴PYY, His³⁴PYY, Lys²⁵hPYY(5-36),Arg⁴hPYY(4-36), Gln⁴hPYY(4-36), Asn⁴hPYY(4-36), Lys²⁵hPYY(4-36),Leu³hPYY(3-36), Val³hPYY(3-36), Lys²⁵hPYY(3-36), Tyr^(1,36)pPYY,Pro¹³Ala¹⁴hPYY, Leu³¹Pro³⁴PYY, FMS-PYY, FMS-PYY(3-36), Fmoc-PYY,Fmoc-PYY(3-36), FMS₂-PYY, FMS₂PYY(3-36), Fmoc₂-PYY, Fmoc₂-PYY(3-36),hPP(1-7)-pNPY, hPP(1-17)-pNPY, hPP(19-23)-pNPY, hPP(19-23)-Pro³⁴pNPY,hPP(19-23)-His³⁴pNPY, rPP(19-23)-pNPY, rPP(19-23)-Pro³⁴pNPY,rPP(19-23)-His³⁴pNPY, hPP(1-7)-pNPY, hPP(1-17)-pNPY,hPP(1-17)-His³⁴pNPY, pNPY(1-7)-hPP, pNPY(1-7, 19-23)-hPP,cPP(1-17)-pNPY(19-23)-hPP, cPP(1-7)-NPY(19-23)-His³⁴hPP,hPP(1-17)-His³⁴pNPY, hPP(19-23)-pNPY, hPP(19-23)-Pro³⁴pNPY,hPP(19-23)-His³⁴pNPY, rPP(19-23)-pNPY, rPP(19-23)-Pro³⁴pNPY,rPP(19-23)-His³⁴pNPY, pNPY(1-7)-hPP, pNPY(19-23)-hPP,pNPY(19-23)-Gln³⁴hPP, pNPY(19-23)-His³⁴hPP, pNPY(19-23)-Phe⁶Gln³⁴hPP,pNPY(19-23)-Phe⁶His³⁴hPP, pNPY(1-7, 19-23)-hPP, pNPY(1-7,19-23)-Gln³⁴hPP, cPP(20-23)-Pro³⁴pNPY, cPP(21-23)-Pro³³-pNPY,cPP(22-23)-Pro³⁴-pNPY, cPP(1-7)-Pro³⁴-pNPY, cPP(20-23)-Pro³⁴-pNPY,cPP(1-7, 20-23)-Pro³⁴-pNPY, cPP(1-7)-pNPY(19-23)-hPP,cPP(1-7)-pNPY(19-23)-His³⁴hPP, or cPP(1-7)-gPP(19-23)-hPP.

In another embodiment, such PPF polypeptides of the invention also donot include: Thr²⁷hPYY(3-36), Ile³⁰hPYY(3-36), Ser³²hPYY(3-36),Lys³³hPYY(3-36), Asn³⁴hPYY(3-36), Lys³⁵hPYY(3-36), Thr³⁶hPYY(3-36),Lys²⁵Thr²⁷hPYY(3-36), Lys²⁵Ile³⁰hPYY(3-36), Lys²⁵Ser³²hPYY(3-36),Lys²⁵Lys³³hPYY(3-36), Lys²⁵Asn²⁴hPYY(3-36), Lys²⁵Lys³³hPYY(3-36),Lys²⁵Thr³⁶PYY(3-36), Thr²⁷Ile²⁸hPYY(3-36), Thr²⁷Val²⁸hPYY(3-36),Thr²⁷Gln²⁹hPYY(3-36), Thr²⁷Ile³⁰hPYY(3-36), Thr²⁷Val³⁰hPYY(3-36),Thr²⁷Ile³¹hPYY(3-36), Thr²⁷Leu³¹hPYY(3-36), Thr²⁷Ser³²hPYY(3-36),Thr²⁷Lys³³hPYY(3-36), Thr²⁷Asn³⁴hPYY(3-36), Thr²⁷Lys³⁵hPYY(3-36),Thr²⁷Thr³⁶hPYY(3-36), Thr²⁷Phe³⁶hPYY(3-36), Phe²⁷Ile³⁰hPYY(3-36),Phe²⁷Ser³²hPYY(3-36), Phe²⁷Lys³³hPYY(3-36), Phe²⁷Asn³⁴hPYY(3-36),Phe²⁷Lys³⁵hPYY(3-36), Phe²⁷Thr³⁶hPYY(3-36), Gln²⁹Ile³⁰hPYY(3-36),Gln²⁹Ser³²hPYY(3-36), Gln²⁹Leu³³hPYY(3-36), Gln²⁹Asn³⁴hPYY(3-36),Gln²⁹Leu³⁵hPYY(3-36), Gln²⁹Thr³⁶hPYY(3-36), Ile³⁰Ile³¹hPYY(3-36),Ile³⁰Leu³¹hPYY(3-36), Ile³⁰Ser³²hPYY(3-36), Ile³⁰Lys³³hPYY(3-36),Ile³⁰Asn³⁴hPYY(3-36), Ile³⁰Lys³⁵hPYY(3-36), Ile³⁰Thr³⁶hPYY(3-36),Ile³⁰Phe³⁶hPYY(3-36), Val³⁰Ser³²hPYY(3-36), Val³⁰Lys³³hPYY(3-36),Val³⁰Asn³⁴hPYY(3-36), Val³⁰Lys³⁵hPYY(3-36), Val³⁰Thr³⁶hPYY(3-36),Ile³¹Ser³²hPYY(3-36), Ile³¹Lys³³hPYY(3-36), Ile³¹Asn³⁴hPYY(3-36),Ile³¹Lys³⁵hPYY(3-36), Ile³¹Thr³⁶hPYY(3-36), Ile³¹Phe³⁶hPYY(3-36),Leu³¹Ser³²hPYY(3-36), Leu³¹Lys³³hPYY(3-36), Leu³¹Asn³⁴hPYY(3-36),Leu³¹Lys³⁵hPYY(3-36), Leu³¹Thr³⁶hPYY(3-36), Ser³²Lys³³hPYY(3-36),Ser³²Asn³⁴hPYY(3-36), Ser³²Lys³⁵hPYY(3-36), Ser³²Thr³⁶hPYY(3-36),Ser³²Phe³⁶hPYY(3-36), Lys³³Asn³⁴hPYY(3-36), Lys³³Lys³⁵hPYY(3-36),Lys³³Thr³⁶hPYY(3-36), Lys³³Phe³⁶hPYY(3-36), Asn³⁴Lys³⁵hPYY(3-36),Asn³⁴Phe³⁶hPYY(3-36), Lys³⁵Thr³⁶hPYY(3-36), Lys³⁵Phe³⁶hPYY(3-36),Thr²⁷hPYY(4-36), Phe²⁷hPYY(4-36), Ile²⁸hPYY(4-36), Val²⁸hPYY(4-36),Gln²⁹hPYY(4-36), Ile³⁰hPYY(4-36), Val³⁰hPYY(4-36), Ile³¹hPYY(4-36),Leu³¹hPYY(4-36), Ser³²hPYY(4-36), Lys³³hPYY(4-36), Asn³⁴hPYY(4-36),Lys³⁵hPYY(4-36), Thr³⁶hPYY(4-36), Phe³⁶hPYY(4-36), Lys²⁵Thr²⁷hPYY(4-36),Lys²⁵Phe²⁷hPYY(4-36), Lys²⁵Ile²⁸hPYY(4-36), Lys²⁵Val²⁸hPYY(4-36),Lys²⁵Gln²⁹hPYY(4-36), Lys²⁵Ile³⁰hPYY(4-36), Lys²⁵Val³⁰hPYY(4-36),Lys²⁵Ile³¹hPYY(4-36), Lys²⁵Leu³¹hPYY(4-36), Lys²⁵Ser³²hPYY(4-36),Lys²⁵Lys³³hPYY(4-36), Lys²⁵Asn²⁴hPYY(4-36), Lys²⁵Lys³⁵hPYY(4-36),Lys²⁵Thr³⁶hPYY(4-36), Lys²⁵Phe³⁶hPYY(4-36), Thr²⁷Ile²⁸hPYY(4-36),Thr²⁷Val²⁸hPYY(4-36), Thr²⁷Gln²⁹hPYY(4-36), Thr²²Ile³⁰hPYY(4-36),Thr²⁷Val³⁰hPYY(4-36), Thr²⁷Ile³¹hPYY(4-36), Thr²⁷Leu³¹hPYY(4-36),Thr²⁷Ser³²hPYY(4-36), Thr²⁷Lys³³hPYY(4-36), Thr²⁷Asn³⁴hPYY(4-36),Thr²⁷Lys³⁵hPYY(4-36), Thr²⁷Thr³⁶hPYY(4-36), Thr²⁷Phe³⁶hPYY(4-36),Phe²⁷Ile²⁸hPYY(4-36), Phe²⁷Val²⁸hPYY(4-36), Phe²⁷Gln²⁹hPYY(4-36),Phe²⁷Ile³⁰hPYY(4-36), Phe²⁷Val³⁰hPYY(4-36), Phe²⁷Ile³¹hPYY(4-36),Phe²⁷Leu³¹hPYY(4-36), Phe²⁷Ser³²hPYY(4-36), Phe²⁷Lys³³hPYY(4-36),Phe²⁷Asn³⁴hPYY(4-36), Phe²⁷Lys³⁵hPYY(4-36), Phe²⁷Thr³⁶hPYY(4-36),Phe²⁷Phe³⁶hPYY(4-36), Gln²⁹Ile³⁰hPYY(4-36), Gln²⁹Val³⁰hPYY(4-36),Gln²⁹Ile³¹hPYY(4-36), Gln²⁹Leu³¹hPYY(4-36), Gln²⁹Ser³², hPYY(4-36)Gln²⁹Leu³³hPYY(4-36), Gln²⁹Asn³⁴hPYY(4-36), Gln²⁹Leu³⁵hPYY(4-36),Gln²⁰Thr³⁶hPYY(4-36), Gln²⁹Phe³⁶hPYY(4-36), Ile³⁰Ile³¹hPYY(4-36),Ile³⁰Leu³¹hPYY(4-36), Ile³⁰Ser³²hPYY(4-36), Ile³⁰Lys³³hPYY(4-36),Ile³⁰Asn³⁴hPYY(4-36), Ile³⁰Lys³⁵hPYY(4-36), Ile³⁰Thr³⁶hPYY(4-36),Ile³⁰Phe³⁶hPYY(4-36), Val³⁰Ile³¹hPYY(4-36), Val³⁰Leu³¹hPYY(4-36),Val³⁰Ser³²hPYY(4-36), Val³⁰Lys³³hPYY(4-36), Val³⁰Asn³⁴hPYY(4-36),Val³⁰Lys³⁵hPYY(4-36), Val³⁰Thr³⁶hPYY(4-36), Val³⁰Phe³⁶hPYY(4-36),Ile³¹Ser³²hPYY(4-36), Ile³¹Lys³³hPYY(4-36), Ile³¹Asn³⁴hPYY(4-36),Ile³¹Lys³⁵hPYY(4-36), Ile³¹Thr³⁶hPYY(4-36), Leu³¹Phe³⁶hPYY(4-36),Leu³¹Phe³⁶hPYY(4-36), Leu³¹Ser³²hPYY(4-36), Val³¹Lys³³hPYY(4-36),Leu³¹Asn³⁴hPYY(4-36), Leu³¹Lys³⁵hPYY(4-36), Leu³¹Thr³⁶hPYY(4-36),Leu³¹Phe³⁶hPYY(4-36), Ser³²Lys³³hPYY(4-36), Ser³²Asn³⁴hPYY(4-36),Ser³²Lys³⁵hPYY(4-36), Ser³²Thr³⁶hPYY(4-36), Ser³²Phe³⁶hPYY(4-36),Lys³³Asn³⁴hPYY(4-36), Lys³³Lys³⁵hPYY(4-36), Lys³³Thr³⁶hPYY(4-36),Lys³³Phe³⁶hPYY(4-36), Asn³⁴Lys³⁵hPYY(4-36), Asn³⁴Phe³⁶hPYY(4-36),Lys³⁵Thr³⁶hPYY(4-36), Lys³⁵Phe³⁶hPYY(4-36), Thr²⁷hPYY(5-36),Phe²⁷hPYY(5-36), Ile²⁸PYY(5-36), Val²⁸hPYY(5-36), Gln²⁹hPYY(5-36),Ile³⁰hPYY(5-36), Val³⁰hPYY(5-36), Ile³¹hPYY(5-36), Leu³¹hPYY(5-36),Ser³²hPYY(5-36), Lys³³hPYY(5-36), Asn³⁴hPYY(5-36), Lys³⁵hPYY(5-36),Thr³⁶hPYY(5-36), Phe³⁶PYY(5-36), Lys²⁵Thr²⁷hPYY(5-36),Lys²⁵Phe²⁷hPYY(5-36), Lys²⁵Ile²⁸hPYY(5-36), Lys²⁵Val²⁸hPYY(5-36),Lys²⁵Gln²⁹hPYY(5-36), Lys²⁵Ile³⁰hPYY(5-36), Lys²⁵Val³⁰hPYY(5-36),Lys²⁵Ile³¹hPYY(5-36), Lys²⁵Leu³¹hPYY(5-36), Lys²⁵Ser³²hPYY(5-36),Lys²⁵Lys³³hPYY(5-36), Lys²⁵Asn²⁴hPYY(5-36), Lys²⁵Lys³⁵hPYY(5-36),Lys²⁵Thr³⁶hPYY(5-36), Lys²⁵Phe³⁶hPYY(5-36), Thr²⁷Ile²⁸hPYY(5-36),Thr²⁷Val²⁸hPYY(5-36), Thr²⁷Gln²⁹hPYY(5-36), Thr²⁷Ile³⁰hPYY(5-36),Thr²⁷Val³⁰hPYY(5-36), Thr²⁷Ile³¹hPYY(5-36), Thr²⁷Leu³¹hPYY(5-36),Thr²⁷Ser³²hPYY(5-36), Thr²⁷Lys³³hPYY(5-36), Thr²⁷Asn³⁴hPYY(5-36),Thr²⁷Lys³⁵hPYY(5-36), Thr²⁷Thr³⁶hPYY(5-36), Thr²⁷Phe³⁶hPYY(5-36),Phe²⁷Ile²⁸hPYY(5-36), Phe²⁷Val²⁸hPYY(5-36), Phe²⁷Gln²⁹hPYY(5-36),Phe²⁷Ile³⁰hPYY(5-36), Phe²⁷Val³⁰hPYY(5-36), Phe²⁷Ile³¹hPYY(5-36),Phe²⁷Leu³¹hPYY(5-36), Phe²⁷Ser³²hPYY(5-36), Phe²⁷Lys³³hPYY(5-36),Phe²⁷Asn³⁴hPYY(5-36), Phe²⁷Lys³⁵hPYY(5-36), Phe²⁷Thr³⁶hPYY(5-36),Phe²⁷Phe³⁶hPYY(5-36), Gln²⁹Ile³⁰hPYY(5-36), Gln²⁹Val³⁰hPYY(5-36),Gln²⁹Ile³¹hPYY(5-36), Gln²⁹Leu³¹hPYY(5-36), Gln²⁹Ser³²hPYY(5-36)Gln²⁹Leu³³hPYY(5-36), Gln²⁹Asn³⁴hPYY(5-36), Gln²⁹Leu³⁵hPYY(5-36),Gln²⁹Thr³⁶hPYY(5-36), Gln²⁹Phe³⁶hPYY(5-36), Ile³⁰Ile³¹hPYY(5-36),Ile³⁰Leu³¹hPYY(5-36), Ile³⁰Ser³²hPYY(5-36), Ile³⁰Lys³³hPYY(5-36),Ile³⁰Asn³⁴hPYY(5-36), Ile³⁰Lys³⁵hPYY(5-36), Ile³⁰Thr³⁶hPYY(5-36),Ile³⁰Phe³⁶hPYY(5-36), Val³⁰Ile³¹hPYY(5-36), Val³⁰Leu³¹hPYY(5-36),Val³⁰Ser³²hPYY(5-36), Val³⁰Lys³³hPYY(5-36), Val³⁰Asn³⁴hPYY(5-36),Val³⁰Lys³⁵hPYY(5-36), Val³⁰Thr³⁶hPYY(5-36), Val³⁰Phe³⁶hPYY(5-36),Ile³¹Ser³²hPYY(5-36), Ile³¹Lys³³hPYY(5-36), Ile³¹Asn³⁴hPYY(5-36),Ile³¹Lys³⁵hPYY(5-36), Ile³¹Thr³⁶hPYY(5-36), Leu³¹Phe³⁶hPYY(5-36),Leu³¹Phe³⁶hPYY(5-36), Leu³¹Ser³²hPYY(5-36), Val³¹Lys³³hPYY(5-36),Leu³¹Asn³⁴hPYY(5-36), Leu³¹Lys³⁵hPYY(5-36), Leu³¹Thr³⁶hPYY(5-36),Leu³¹Phe³⁶hPYY(5-36), Ser³²Lys³³hPYY(5-36), Ser³²Asn³⁴hPYY(5-36),Ser³²Lys³⁵hPYY(5-36), Ser³²Thr³⁶hPYY(5-36), Ser³²Phe³⁶hPYY(5-36),Lys³³Asn³⁴hPYY(5-36), Lys³³Lys³⁵hPYY(5-36), Lys³³Thr³⁶hPYY(5-36),Lys³³Phe³⁶hPYY(5-36), Asn³⁴Lys³⁵hPYY(5-36), Asn³⁴Phe³⁶hPYY(5-36),Lys³⁵Thr³⁶hPYY(5-36), or Lys³⁵Phe³⁶hPYY(5-36).

In another embodiment, the PPF polypeptides of the invention do notinclude any unnatural amino acid residues, and comprise a C-terminaltail motif of hPYY. The C-terminal tail motif may comprise amino acidresidues 32-35 of hPYY, e.g., Thr, Arg, Gln, Arg (SEQ ID NO: 351). Insuch an embodiment, the PPF polypeptides of the invention do not includeany native PPF polypeptides (e.g., NPY(1-36), NPY(3-36), PYY(1-36),PYY(3-36)), NPY(2-36), PYY(4-36), PYY(5-36)), (2-36)NPY, (2-36)PYY,Gln³⁴hPP, Ile³¹Gln³⁴PP, Ala¹NPY, Tyr¹NPY, Ala²NPY, Leu²NPY, Phe²NPY,His²NPY, Ala³NPY, Ala⁴NPY, Ala⁶NPY, Tyr⁷pNPY, Ala⁷NPY, Ala⁹NPY,Ala¹⁰NPY, Ala¹¹NPY, Gly¹²NPY, Ala¹³NPY, Gly¹⁴NPY, Ala¹⁵NPY, Ala¹⁶NPY,Ala¹⁷NPY, Gly¹⁸NPY Ala¹⁹NPY, Lys¹⁹NPY, Ala²⁰NPY, Ala²¹NPY, Ala²²NPY,Gly²³NPY, Ala²⁴NPY, Trp²⁴pNPY, Ala²⁵NPY, Lys²⁵NPY, Ala²⁶NPY, Ala²⁷NPY,Phe²⁷NPY, Ala²⁸NPY, Ala²⁹NPY, Gln²⁹NPY, Ala³⁰NPY, Phe³⁰NPY, Ala³¹NPY,Trp³¹pNPY, Ala³⁶NPY, Phe³⁶NPY, His³⁶NPY, Ahx(9-22)pNPY, Ahx(9-17)pNPY,des-AA(10-17)-pNPY, des-AA(10-17)-Cys^(2,27)-pNPYdes-AA(10-17)-Ala^(2,21)-pNPY, des-AA(10-17)-Cys^(7,21)-pNPY,des-AA(10-17)-Glu⁷Lys²¹-pNPY, Lys²⁵hPYY(5-36), Arg⁴hPYY(4-36),Gln⁴hPYY(4-36), Asn⁴hPYY(4-36), Lys²⁵hPYY(4-36), Leu³hPYY(3-36),Val³hPYY(3-36), Lys²⁵hPYY(3-36), Tyr^(1,36)pPYY, Pro¹³Ala¹⁴hPYY,FMS-PYY, FMS-PYY(3-36), Fmoc-PYY, Fmoc-PYY(3-36), FMS₂-PYY,FMS₂-PYY(3-36), Fmoc₂-PYY, Fmoc₂-PYY(3-36), hPP(1-7)-pNPY,hPP(1-17)-pNPY, hPP(19-23)-pNPY, rPP(19-23)-pNPY, hPP(1-7)-pNPY,hPP(1-17)-pNPY, hPP(19-23)-pNPY, rPP(19-23)-pNPY, pNPY(19-23)-Gln³⁴hPP,pNPY(19-23)-Phe⁶Gln³⁴hPP, or pNPY(1-7, 19-23)-Gln³⁴hPP.

In another aspect, such PPF polypeptides of the invention comprising aC-terminal tail motif of hPYY also do not include: Thr²⁷hPYY(3-36),Ile³⁰hPYY(3-36), Thr³⁶hPYY(3-36), Lys²⁵Thr²⁷hPYY(3-36),Lys²⁵Ile³⁰hPYY(3-36), Lys²⁵Asn²⁴hPYY(3-36), Lys²⁵Thr³⁶hPYY(3-36),Thr²⁷Ile²⁸hPYY(3-36), Thr²⁷Val²⁸hPYY(3-36), Thr²⁷Gln²⁹hPYY(3-36),Thr²⁷Ile³⁰hPYY(3-36), Thr²⁷Val³⁰hPYY(3-36), Thr²⁷Ile³¹hPYY(3-36),Thr²⁷Leu³¹hPYY(3-36), Thr²⁷Thr³⁶hPYY(3-36), Thr²⁷Phe³⁶hPYY(3-36),Phe²⁷Ile³⁰hPYY(3-36), Phe²⁷Thr³⁶hPYY(3-36), Gln²⁹Ile³⁰hPYY(3-36),Gln²⁹Thr³⁶hPYY(3-36), Ile³⁰Ile³¹hPYY(3-36), Ile³⁰Leu³¹hPYY(3-36),Ile³⁰Thr³⁶hPYY(3-36), Ile³⁰Phe³⁶hPYY(3-36), Val³⁰Thr³⁶hPYY(3-36),Ile³¹Thr³⁶hPYY(3-36), Ile³¹Phe³⁶hPYY(3-36), Leu³¹Thr³⁶hPYY(3-36),Thr²⁷hPYY(4-36), Phe²⁷hPYY(4-36), Ile²⁸hPYY(4-36), Val²⁸hPYY(4-36),Gln²⁹hPYY(4-36), Phe³⁰hPYY(4-36), Val³⁰hPYY(4-36), Ile³¹hPYY(4-36),Leu³¹hPYY(4-36), Thr³⁶hPYY(4-36), Phe³⁶hPYY(4-36), Lys²⁵Thr²⁷hPYY(4-36),Lys²⁵Phe²⁷hPYY(4-36), Lys²⁵Ile²⁸hPYY(4-36), Lys²⁵Val²⁸hPYY(4-36),Lys²⁵Gln²⁹hPYY(4-36), Lys²⁸Ile³⁰hPYY(4-36), Lys²⁵Val³⁰hPYY(4-36),Lys²⁵Ile³¹hPYY(4-36), Lys²⁵Leu³¹hPYY(4-36), Lys²⁵Thr³⁶hPYY(4-36),Lys²⁵Phe³⁶hPYY(4-36), Thr²⁷Ile²⁸hPYY(4-36), Thr²⁷Val²⁸hPYY(4-36),Thr²⁷Gln²⁹hPYY(4-36), Thr²⁷Ile³⁰hPYY(4-36), Thr²⁷Val³⁰hPYY(4-36),Thr²⁷Ile³¹hPYY(4-36), Thr²⁷Leu³¹hPYY(4-36), Thr²⁷Thr³⁶hPYY(4-36),Thr²⁷Phe³⁶hPYY(4-36), Phe⁷¹Ile²⁸hPYY(4-36), Phe²⁷Val²⁸hPYY(4-36),Phe²⁷Gln²⁹hPYY(4-36), Phe²⁷Ile³⁰hPYY(4-36), Phe²⁷Val³⁰hPYY(4-36),Phe²⁷Ile³¹hPYY(4-36), Phe²⁷Leu³¹hPYY(4-36), Phe²⁷Thr³⁶hPYY(4-36),Phe²⁷Phe³⁶hPYY(4-36), Gln²⁹Ile³⁰hPYY(4-36), Gln²⁹Val³⁰hPYY(4-36),Gln²⁹Ile³¹hPYY(4-36), Gln²⁹Leu³¹hPYY(4-36), Gln²⁹Thr³⁶hPYY(4-36),Gln²⁹Phe³⁶hPYY(4-36), Ile³⁰Ile³¹hPYY(4-36), Ile³⁰Leu³¹hPYY(4-36),Ile³⁰Thr³⁶hPYY(4-36), Ile³⁰Phe³⁶hPYY(4-36), Val³⁰Ile³¹hPYY(4-36),Val³⁰Leu³¹hPYY(4-36), Val³⁰Thr³⁶hPYY(4-36), Val³⁰Phe³⁶hPYY(4-36),Ile³¹Thr³⁶hPYY(4-36), Leu³¹Phe³⁶hPYY(4-36), Leu³¹Phe³⁶hPYY(4-36),Leu³³Thr³⁶hPYY(4-36), Leu³⁴Phe³⁶hPYY(4-36), Thr²⁷hPYY(5-36),Phe²⁷hPYY(5-36), Ile²⁸hPYY(5-36), Val²⁸hPYY(5-36), Gln²⁹hPYY(5-36),Ile³⁰hPYY(5-36), Val³⁰hPYY(5-36), Ile³¹hPYY(5-36), Leu³¹hPYY(5-36),Thr³⁶hPYY(5-36), Phe³⁶hPYY(5-36), Lys²⁵Thr²⁷hPYY(5-36),Lys²⁵Phe²⁷hPYY(5-36), Lys²⁵Ile²⁸hPYY(5-36), Lys²⁵Val²⁸hPYY(5-36),Lys²⁵Gln²⁹hPYY(5-36), Lys²⁵Ile³⁰hPYY(5-36), Lys²⁵Val³⁰hPYY(5-36),Lys²⁵Ile³¹hPYY(5-36), Lys²⁵Leu³¹hPYY(5-36), Lys²⁵Thr³⁶hPYY(5-36),Lys²⁵Phe³⁶hPYY(5-36), Thr²⁷Ile²⁸hPYY(5-36), Thr²⁷Val²⁸hPYY(5-36),Thr²⁷Gln²⁹hPYY(5-36), Thr²⁷Ile³⁰hPYY(5-36), Thr²⁷Val³⁰hPYY(5-36),Thr²⁷Ile³¹hPYY(5-36), Thr²⁷Leu³¹hPYY(5-36), Thr²⁷Thr³⁶hPYY(5-36),Thr²⁷Phe³⁶hPYY(5-36), Phe²⁷Ile²⁸hPYY(5-36), Phe²⁷Val²⁸hPYY(5-36),Phe²⁷Gln²⁹hPYY(5-36), Phe²⁷Ile³⁰hPYY(5-36), Phe²⁷Val³⁰hPYY(5-36),Phe²⁷Ile³¹hPYY(5-36), Phe²⁷Leu³¹hPYY(5-36), Phe²⁷Thr³⁶hPYY(5-36),Phe²⁷Phe³⁶hPYY(5-36), Gln²⁹Ile³⁰hPYY(5-36), Gln²⁹Val³⁰hPYY(5-36),Gln²⁹Ile³¹hPYY(5-36), Gln²⁹Leu³¹hPYY(5-36), Gln²⁹Thr³⁶hPYY (5-36),Gln²⁹Phe³⁶hPYY(5-36), Ile³⁰Ile³¹hPYY(5-36), Ile³⁰Leu³¹hPYY(5-36),Ile³⁰Thr³⁶hPYY(5-36), Ile³⁰Phe³⁶hPYY(5-36), Val³⁰Ile³¹hPYY(5-36),Val³⁰Leu³¹hPYY(5-36), Val³⁰Thr³⁶hPYY(5-36), Val³⁰Phe³⁶hPYY(5-36),Ile³¹Thr³⁶hPYY(5-36), Leu³¹Phe³⁶hPYY(5-36), Leu³¹Phe³⁶hPYY(5-36),Leu³¹Thr³⁶hPYY(5-36), Leu³¹Phe³⁶hPYY(5-36).

In yet another embodiment, the PPF polypeptides of the invention do notinclude those PPF-related polypeptides disclosed in WO 03/026591 and WO03/057235, which are herein incorporated by reference in their entirety.

In another embodiment, the polypeptides of the invention are at least 34amino acids in length. In other embodiments, the PPF polypeptides may beat least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 aminoacids in length. Further, in one embodiment, the polypeptides of theinvention include only natural L amino acid residues and/or modifiednatural L amino acid residues. Alternatively, in another embodiment, thepolypeptides of the invention do not include unnatural amino acidresidues.

In yet another embodiment, PPF polypeptides of the invention may exhibitat least 60%, 65%, 70%, 80%, or 90% sequence identity to a nativePYY(3-36) over the entire length of the PYY(3-36). Such PPF polypeptidesof the invention may also exhibit at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 92%, at least 94% or at least97% sequence identity to a native PP. In yet another embodiment, suchPPF polypeptides of the invention may exhibit at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 92%, at least94% or at least 97% sequence identity to a native NPY.

More specifically, in one aspect, the present invention relates to novelPPF polypeptides comprising at least two PPF motifs, wherein the atleast two PPF motifs include at least the N-terminal polyproline PPFmotif and the C-terminal tail PPF motif, and the PPF polypeptide doesnot include any unnatural amino acid residues. Such PPF polypeptides ofthe invention will exhibit at least 50% sequence identity to a nativePYY(3-36) over the entire length of the PYY(3-36). In some embodiments,such PPF polypeptides have at least 34 amino acid residues. In someembodiments, such PPF polypeptides of the invention may exhibit at least60%, at least 70%, at least 80%, at least 90%, at least 92%, at least94% or at least 97% sequence identity to a native PYY(3-36) over theentire length of the PYY(3-36). Such PPF polypeptides of the inventionmay also exhibit at least 50%, at least 60%, at least 70%, at least 80%,at least 90%, at least 92%, at least 94% or at least 97% sequenceidentity to a native PP. In some embodiments, the PPF polypeptides maycomprise not more than 10, not more than 5, not more than 3, not morethan 2 or not more than 1 amino acid substitutions. In yet anotherembodiment, such PPF polypeptides of the invention may exhibit at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least92%, at least 94% or at least 97% sequence identity to a native NPY.

In another aspect, the PPF polypeptides of the invention include PYYanalog polypeptides. In yet another aspect of the invention, the PPFpolypeptides of the invention include PPF chimeric polypeptidescomprising a fragment of a PP, PYY or NPY polypeptide covalently linkedto at least one additional fragment of a PP, PYY or NPY polypeptide,wherein each PP, PYY or NPY fragment includes a PPF motif. Such PPFanalog polypeptides and PPF chimeric polypeptides of the invention willexhibit at least 50% sequence identity to a native PYY(3-36) over theentire length of the PYY(3-36). In some embodiments, such PPFpolypeptides of the invention may exhibit at least 60%, at least 70%, atleast 80%, at least 90%, at least 92%, at least 94% or at least 97%sequence identity to a native PYY(3-36) over the entire length of thePYY(3-36). PPF polypeptides of the invention may also exhibit at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least92%, at least 94% or at least 97% sequence identity to a native PP. Inyet another embodiment, PPF polypeptides of the invention may exhibit atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 92%, at least 94% or at least 97% sequence identity to a nativeNPY. In certain embodiments, desirable PPF polypeptides may not includeN-terminal PP fragments in combination with C-terminal NPY fragments.

PPF polypeptides useful in the invention may have a PPF activity greaterthan or less than the native compounds for a particular activity. Thus,for example, PYY agonists may have 3, 5, 10, 50, 100, 500, 1000 times ormore activity than native PYY. Furthermore, while it is desirable to usea PYY agonist having similar or greater activity than native PYY, one ofordinary skill in the art would understand that agonists having lessactivity than native PYY would also be useful in the present invention.Such agonists, for example, may have anywhere from 2, 5, 10, 15, or 20times less activity than native PYY, but possess other desirablecharacteristics, e.g., solubility, bioavailability, ease inmanufacturing or formulation, or fewer side effects. In someembodiments, a PPF polypeptide useful in the invention may be a PYYantagonist.

Examples of PYY agonists, more particularly PYY analog agonists oragonist analogs (analogs and derivatives of PYY), are described in U.S.Pat. No. 5,574,010, WO04/089279, WO 04/066966, WO 03/057235, WO03/026591, WO 98/20885, WO 94/22467, the contents of which areincorporated by reference in their entirety. Other PYY analog agonistsare described in Balasubramaniam et al., Pept Res 1(1):32-5, 1998,Balasubramaniam et al., Peptides 14: 1011-1016, 1993, Boublik et al., J.Med. Chem. 32: 597-601, 1989, Liu et al., J. Gastrointest Surg5(2):147-52, 2001. Gehlert et al., Proc Soc Exp Biol Med, 218:7-22,1998, Sheikh et al., Am J Physiol. 261:G701-15, 1991, Potter et al., EurJ Pharmacol 267(3): 253-362, 1994, Lebon et al., J. Med. Chem.38:1150-57, 1995, Fournier et al., Mol Pharmacol 45(1):93-101, 1994,Kirby et al., J. Med Chem 38:4579-86, 1995, Beck et al., FEBS Letters244(1): 119-122, 1989, Rist et al., Eur J Biochemistry 247: 1019-1028,1997, Soll et al., Eur J Biochem 268 (10): 2828-37, 2001, Cabrele etal., J Pept Sci 6(3): 97-122, 2000, Balasubramaniam et al., J Med Chem43: 3420-3427, 2000, Kirby et al., J Med Chem 36:3802-08, 1993,Grundemar et al., Regulatory Peptides 62:131-136, 1996, Feinstein et al.J Med Chem 35:2836-2843, 1992, Cox et al. Regulatory Peptides 75-76:3-8, 1998, Cabrele et al., Peptides 22: 365-378, 2001, Keire et al.Biochemistry 39: 9935-9942, 2000, Keire et al. Am. J. PhysiolGastrointest Liver Physiol 279: G126-G131, 2000, and incorporated hereinby reference.

PYY, NPY, and PP constitute a family of C-terminally amidated peptidesinvolved in the regulation of gastrointestinal function, blood pressure,and feeding behavior. Without intending to be limited by theory, theability of these peptides to selectively bind and activate Y receptorsubtypes is believed to depend strongly on a stable solution structure,including the so-called “PP-fold”. Table 1 (below) shows PP familyligand potencies at the known receptors and the rank order of potenciesof various ligands.

TABLE 1 Summary of receptor pharmacology for the PP family of receptorsRECEPTORS PHARMACOLOGY REFERENCE Food Intake PYY(3-36) ≧ PYY >> NPY,NPY(3-36), Inhibition PP, Ac-PYY(22-36) (peripheral) Gastric PYY(3-36) ≧PYY >> NPY, NPY(3-36), Emptying PP, Ac-PYY(22-36) Food Intake PYY ≧PYY(3-36) = NPY = NPY(3-36) > Iyengar et al., J. Pharmacol. Exp. Ther.289: 1031-40, Stimulation PP 1999 (central) Y1 NPY = PYY > NPY(3-36) =PYY(3-36) = Iyengar et al., J. Pharmacol. Exp. Ther. 289: 1031-40, PP1999; Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22, 1998; Michel etal., Pharmacol. Rev. 50: 143-50, 1998; U.S. Pat. No. 5,968,819 Y2 NPY =PYY = PYY(3-36) = NPY(3-36) >> Iyengar et al., J. Pharmacol. Exp. Ther.289: 1031-40, PP 1999; Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22,1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998; U.S. Pat. No.5,968,819 Y3 NPY > PP > PYY Gehlert, Proc. Soc. Exp. Biol. Med. 218:7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998. Y4 PP >PYY > NPY > PYY(3-36) = Iyengar et al., J. Pharmacol. Exp. Ther. 289:1031-40, NPY(3-36) 1999; Gehlert, Proc. Soc. Exp. Biol. Med. 218: 7-22,1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998; U.S. Pat. No.5,968,819 Y5 NPY = PYY ≧ PP ≧ PYY(3-36) = Iyengar et al., J. Pharmacol.Exp. Ther. 289: 1031-40, NPY(3-36) 1999; Gehlert, Proc. Soc. Exp. Biol.Med. 218: 7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998;U.S. Pat. No. 5,968,819 Y6 NPY = PYY ≧ NPY(3-36) > PP Iyengar et al., J.Pharmacol. Exp. Ther. 289: 1031-40, 1999; Gehlert, Proc. Soc. Exp. Biol.Med. 218: 7-22, 1998; Michel et al., Pharmacol. Rev. 50: 143-50, 1998;U.S. Pat. No. 5,968,819 (Y7) PYY > NPY >> PYY(3-36) = PP Yang et al.,Br. J. Pharmacol. 123: 1549-54, 1998 (Y7) PYY(3-36) ≧ PYY > NPY >> PPHaynes et al., Br. J. Pharmacol. 122: 1530-6, 1997 (Y7) PYY >> NPY =PYY(3-36) = PP Kawakubo et al., Brain Res. 854: 30-4, 2000

Research has suggested that the differences in Y receptor bindingaffinities are correlated with secondary and tertiary structuraldifferences. See. e.g., Keire et al., Biochemistry 2000, 39, 9935-9942.Native porcine PYY has been characterized as including two C-terminalhelical segments from residues 17 to 22 and 23 to 33 separated by a kinkat residues 23, 24, and 25, a turn centered around residues 12-14, andthe N-terminus folded near residues 30 and 31. Further, full-lengthporcine PYY has been characterized as including the PP fold, stabilizedby hydrophobic interactions among residues in the N- and C-termini. Seeid.

By “PP” is meant a pancreatic peptide polypeptide obtained or derivedfrom any species. Thus, the term “PP” includes both the human fulllength, 36 amino acid peptide as set forth in SEQ ID NO: 1, and speciesvariations of PP, including, e.g., murine, hamster, chicken, bovine,rat, and dog PP. In this sense, “PP,” “wild-type PP,” and “native PP,”i.e., unmodified PP, are used interchangeably.

By “NPY” is meant a neuropeptide Y polypeptide obtained or derived fromany species. Thus, the term “NPY” includes both the human full length,36 amino acid peptide as set forth in SEQ ID NO: 4, and speciesvariations of NPY, including, e.g., murine, hamster, chicken, bovine,rat, and dog NPY. In this sense, “NPY,” “wild-type NPY,” and “nativeNPY”, i.e., unmodified NPY, are used interchangeably.

By “PYY” is meant a peptide YY polypeptide obtained or derived from anyspecies. Thus, the term “PYY” includes both the human full length, 36amino acid peptide as set forth in SEQ ID NO: 2, and species variationsof PYY, including e.g., marine, hamster, chicken, bovine, rat, and dogPYY. In this sense, “PYY” and “wild-type PYY” and “native PYY,” i.e.,unmodified PYY, are used interchangeably. In the context of the presentinvention, all modifications discussed with reference to the PYY analogpolypeptides of the present invention are based on the 36 amino acidsequence of native human PYY (SEQ ID NO: 2).

By “PP agonist”, “PYY agonist”, or “NPY agonist” is meant a compoundwhich elicits a biological activity of native human PP, PYY, or NPY,respectively. In some embodiments, the terms refer to a compound whichelicits a biological effect in the reduction of nutrient availabilitysimilar to that of native human PP, PYY, or NPY, for example a compound(1) having activity in food intake, gastric emptying, pancreaticsecretion, or weight loss assays similar to native human PP, PYY, orNPY, and/or (2) which binds specifically in a Y receptor assay or in acompetitive binding assay with labeled PP, PYY, PYY(3-36), or NPY fromcertain tissues having an abundance of Y receptors, including, e.g.,area postrema. In some embodiments, the agonist is not PP, PYY,PYY(3-36), and/or NPY. In some embodiments, the agonists will bind insuch assays with an affinity of greater than 1 μM. In some embodiments,the agonists will bind in such assays with an affinity of greater than1-5 nM. Such agonists may comprise a polypeptide having a PPF motif, anactive fragment of PP, PYY, or NPY, or a small chemical molecule.

PYY, PYY(3-36) or agonists thereof may be modified at the N-terminalend, C-terminal end and/or along its length to modify othercharacteristics as available in the art. Insertions, extensions, orsubstitutions as described above may be with other natural amino acids,synthetic amino acids, peptidomimetics, or other chemical compounds. Theanalog polypeptides of the invention may be derivatized by chemicalalterations such as amidation, glycosylation, acylation, sulfation,phosphorylation, acetylation, and cyclization. Such chemical alterationsmay be obtained through chemical or biochemical methodologies, as wellas through in-vivo processes, or any combination thereof. Derivatives ofthe analog polypeptides of the invention may also include conjugation toone or more polymers or small molecule substituents. One type of polymerconjugation is linkage or attachment of polyethylene glycol (“PEG”)polymers, polyamino acids (e.g., poly-his, poly-arg, poly-lys, etc.)and/or fatty acid chains of various lengths to the N- or C-terminus oramino acid residue side chains of a polypeptide analog. Small moleculesubstituents include short alkyls and constrained alkyls (e.g.,branched, cyclic, fused, adamantyl), and aromatic groups.

“Reduced nutrient availability” is meant to include any means by whichthe body reduces the nutrients available to the body to store as fat.Reducing nutrient availability may be by means that include, but are notlimited to, reducing appetite, increasing satiety, affecting foodchoice/taste aversion, increasing metabolism, and/or decreasing orinhibiting food absorption. Exemplary mechanisms that may be affectedinclude delayed gastric emptying or decreased absorption of food in theintestines.

“Increased nutrient availability” is meant to include any means by whichthe body increases the nutrients available to the body to store as fat.Increasing nutrient availability may be by means that include, but arenot limited to, increasing appetite, decreasing satiety, affecting foodchoice, decreasing taste aversion, decreasing metabolism, and/orincreasing food absorption. Exemplary mechanisms that may be affectedinclude decreasing gastric hypomotility or increasing absorption of foodin the intestines.

With regard to the methods to reduce nutrient availability, as usedherein, a “subject in need thereof” includes subjects who are overweightor obese or morbidly obese, or desirous of losing weight. In addition,subjects who are insulin resistant, glucose intolerant, or have any formof diabetes mellitus (e.g., type 1, 2 or gestational diabetes) canbenefit from these methods to reduce nutrient availability.

With regard to the methods to increase nutrient availability, as usedherein, a “subject in need thereof” includes subjects who areunderweight or desirous of gaining weight.

A “subject” is meant to include any animal, including humans, primates,and other mammals including rats, mice, pets such as cats, dogs,livestock such as horses, cattle, pigs, sheep and goats, as well aschickens, turkeys and any other commercially valuable or companionanimal for which body weight or altering body composition may be anissue.

As used herein, the term “dieting” is meant to include any means bywhich a subject has a reduced caloric intake relative to his or hercaloric intake before beginning dieting. Examples of dieting mayinclude, but are not limited to, reducing the total amount of foodconsumed overall, reducing consumption of any one or more of protein,carbohydrate or fat components of the diet, or reducing the proportionof fat relative to the proportion of carbohydrates and/or protein in thediet.

By “metabolic rate” is meant the amount of energy liberated/expended perunit of time. Metabolism per unit time can be estimated by foodconsumption, energy released as heat, or oxygen used in metabolicprocesses. It is generally desirable to have a higher metabolic ratewhen one wants to lose weight. For example, a person with a highmetabolic rate may be able to expend more energy (e.g., the body burnsmore calories) to perform an activity than a person with a low metabolicrate for that activity.

As used herein, “lean mass” or “lean body mass” refers to muscle andbone. Lean body mass does not necessarily indicate fat free mass. Leanbody mass contains a small percentage of fat (roughly 3%) within thecentral nervous system (brain and spinal cord), marrow of bones, andinternal organs. Lean body mass is measured in terms of density. Methodsof measuring fat mass and lean mass include, but are not limited to,underwater weighing, air displacement plethysmograph, x-ray, DEXA scans,magnetic resonance imaging (MRI), computed tomography (CT) scans,adiabatic bomb calorimetry. In some embodiment, body composition ismeasured pre- and post treatment using a rodent NMR machine(EchoMRI-700™). Animals are placed in a restraining tube and placed inthe NMR for 2 minutes, and the amount of fat and lean mass, in grams, isquantified. In some embodiments, body composition (lean mass, fat mass)for each animal is analyzed using a Dual Energy X-ray Absorptiometry(DEXA) instrument per manufacturer's instructions (Lunar Piximus, GEImaging System). In some embodiments, fat mass and lean mass is measuredusing underwater weighing. In some embodiments, body composition ismeasured using MRI. In some embodiments, body composition is measuredusing a CT scan. In some embodiments, body composition is measured usingadiabatic bomb calorimetry. In some embodiments, body composition ismeasured using an x-ray. In some embodiments, body composition ismeasured using an air displacement plethysmograph. In some embodiments,at the time of termination of animal subjects, the retroperitoneal andmesenteric fat pads, markers of visceral adiposity, are dissected outand weighed.

By “fat distribution” is meant the location of fat deposits in the body.Such locations of fat deposition include, for example, subcutaneous,visceral and ectopic fat depots.

By “subcutaneous fat” is meant the deposit of lipids just below theskin's surface. The amount of subcutaneous fat in a subject can bemeasured using any method available for the measurement of subcutaneousfat. Methods of measuring subcutaneous fat are known in the art, forexample, those described in U.S. Pat. No. 6,530,886, the entirety ofwhich is incorporated herein by reference.

By “visceral fat” is meant the deposit of fat as intra-abdominal adiposetissue. Visceral fat surrounds vital organs and can be metabolized bythe liver to produce blood cholesterol. Visceral fat has been associatedwith increased risks of conditions such as polycystic ovary syndrome,metabolic syndrome and cardiovascular diseases.

By “ectopic fat storage” is meant lipid deposits within and aroundtissues and organs that constitute the lean body mass (e.g., skeletalmuscle, heart, liver, pancreas, kidneys, blood vessels). Generally,ectopic fat storage is an accumulation of lipids outside classicaladipose tissue depots in the body.

As used herein, and as well-understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. “Treating” or “palliating” a disease, disorder, or conditionmeans that the extent and/or undesirable clinical manifestations of acondition, disorder, or a disease state are lessened and/or time courseof the progression is slowed or lengthened, as compared to not treatingthe disorder. For example, in treating obesity, a decrease in bodyweight, e.g., at least a 5% decrease in body weight, is an example of adesirable treatment result. Beneficial or desired clinical resultsinclude, but are not limited to, alleviation or amelioration of one ormore symptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Further, treating does not necessarily occur byadministration of one dose, but often occurs upon administration of aseries of doses. Thus, a therapeutically effective amount, an amountsufficient to palliate, or an amount sufficient to treat a disease,disorder, or condition may be administered in one or moreadministrations.

As used herein, the term “therapeutically effective amount” means theamount of the PPF polypeptide in the composition that will elicit thebiological or medical response in a tissue, system, subject, or humanthat is being sought by the subject, researcher, veterinarian, medicaldoctor or other clinician, which includes alleviation of the symptoms ofthe disorder being treated. The novel methods of treatment of thisinvention are for disorders known to those skilled in the art.

As used herein, the term “prophylactically effective amount” means theamount of the active compounds in the composition that will elicit thebiological or medical response in a tissue, system, subject, or humanthat is being sought by the subject, researcher, veterinarian, medicaldoctor or other clinician, to prevent the onset of obesity or anobesity-related disorder, condition or disease in subjects as risk forobesity or the obesity-related disorder, condition or disease.

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless otherwise indicated or clear from context. Forexample, as will be apparent from context, “a” PYY agonist can includeone or more PYY agonists.

By “amino acid” and “amino acid residue” is meant natural amino acids,unnatural amino acids, and modified amino acid. Unless stated to thecontrary, any reference to an amino acid, generally or specifically byname, includes reference to both the D and the L stereoisomers if theirstructure allow such stereoisomeric forms. Natural amino acids includealanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp),cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly),histidine (His), isoleucine (Ile), leucine (Leu), Lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).Unnatural amino acids include, but are not limited to homolysine,homoarginine, azetidinecarboxylic acid, 2-aminoadipic acid,3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyricacid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid,tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodemosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid, thioproline,sarcosine and citrulline. Additional unnatural amino acids includemodified amino acid residues which are chemically blocked, reversibly orirreversibly, or chemically modified on their N-terminal amino group ortheir side chain groups, as for example, N-methylated D and L aminoacids or residues wherein the side chain functional groups arechemically modified to another functional group. For example, modifiedamino acids include methionine sulfoxide; methionine sulfone; asparticacid-(beta-methyl ester), a modified amino acid of aspartic acid;N-ethylglycine, a modified amino acid of glycine; or alaninecarboxamide, a modified amino acid of alanine. Additional residues thatcan be incorporated are described in Sandberg et al., J. Med. Chem. 41:2481-91, 1998.

By “Ahx” is meant 6-amino hexanoic acid.

Certain human sequences of peptides in the PPF are as follows (inconventional one-letter amino acid code):

PP: (SEQ ID NO: 1) APLEPVYPGD NATPEQMAQY AADLRRYINM LTRPRY PPY:(SEQ ID NO: 2) YPIKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY PYY(3-36):(SEQ ID NO: 3) IKPEAPGE DASPEELNRY YASLRHYLNL VTRQRY NPY: (SEQ ID NO: 4)YPSKPDNPGE DAPAEDMARY YSALRHYINL ITRQRY

Species homologs of human PYY include those amino acid sequences of SEQID NOs. 7-29.

As mentioned above, these peptides are C-terminally amidated whenexpressed physiologically, but need not be for the purposes of theinstant invention. In other words, the C-terminus of these peptides, aswell as the PPF polypeptides of the present invention, may have a free—OH or —NH₂ group. These peptides may also have other post-translationalmodifications. One skilled in the art will appreciate that the PPFpolypeptides of the present invention may also be constructed with anN-terminal methionine residue.

PPF polypeptides of the invention include the PPF polypeptides of theFormula (I) (SEQ ID NO: 30):

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Pro Xaa₆ Xaa₇ Pro Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈Xaa₁₉ Tyr Xaa₂₁ Xaa₂₂ Xaa₂₃ Leu Xaa₂₅ Xaa₂₆ Xaa₂₇Xaa₂₈ Xaa₂₉ Xaa₃₀ Xaa₃₁ Thr Arg Gln Arg Xaa₃₆wherein:

Xaa₁ is Tyr, Ala, Phe, Trp, or absent;

Xaa₂ is Pro, Gly, d-Ala, homoPro, hydroxyPro, or absent;

Xaa₃ is Ile, Ala, NorVal, Val, Leu, Pro, Ser, Thr or absent;

Xaa₄ is Lys, Ala, Gly, Arg, d-Ala, homoLys, homo-Arg, Glu, Asp, orabsent;

Xaa₆ is Glu, Ala, Val, Asp, Asn, or Gln;

Xaa₇ is Ala, Asn, His, Ser, or Tyr;

Xaa₉ is Gly, Ala Ser, sarcosine, Pro, or Aib;

Xaa₁₀ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₁ is Asp, Ala, Glu, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₂ is Ala or d-Ala;

Xaa₁₃ is Ser, Ala, Thr, Pro, or homoSer;

Xaa₁₄ is Pro, Ala, homo-Pro, hydroxyPro, Aib, or Gly;

Xaa₁₅ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₆ is Glu, Ala, Asp, Asn, or Gln;

Xaa₁₇ is Leu, Ala, Met, Trp, Ile, Val, or NorVal;

Xaa₁₈ is Asn, Asp, Ala, Glu, Gln, Ser, or Thr;

Xaa₁₉ is Arg, Tyr, Lys, Ala, Gln, or N(Me)Ala;

Xaa₂₁ is Tyr, Ala, Met, Phe, or Leu;

Xaa₂₂ is Ala, Ser, Thr, or d-Ala;

Xaa₂₃ is Ser, Ala, Asp, Thr, or homoSer;

Xaa₂₅ is Arg, homoArg, Lys, homoLys, Orn, or Cit;

Xaa₂₆ is His, Ala, Arg, homoArg, homoLys, Orn, or Cit;

Xaa₂₇ is Tyr or Phe;

Xaa₂₈ is Leu, Ile, Val, or Ala;

Xaa₂₉ is Asn or Gln;

Xaa₃₀ is Leu, Ala, NorVal, Val, Ile, or Met;

Xaa₃₁ is Ala, Val, Ile, or Leu; and

Xaa₃₆ is Tyr, N(Me)Tyr, His, Trp, or Phe;

with the proviso that said PPF polypeptide is not a native PPFpolypeptide, NPY(2-36), NPY(4-36), PYY(2-36), PYY(4-36), PP(2-36),PP(4-36), Ala¹NPY, Ala³NPY, Ala⁴NPY, Ala⁶NPY, Ala⁷NPY, Tyr⁷pNPY,Ala⁹NPY, Ala¹⁰NPY, Ala¹¹NPY, Ala¹³NPY, Gly¹⁴NPY, Ala¹⁵NPY, Ala¹⁶NPY,Ala¹⁷NPY, Ala¹⁹NPY, Lys¹⁹NPY, Ala²¹NPY, Ala²²NPY, Lys²⁵NPY, Ala²⁶NPY,Phe²⁷NPY, Ala²⁸NPY, Gln²⁹NPY, Ala³⁰NPY, Ala³¹NPY, Phe³⁶NPY, His³⁶NPY,Leu³hPYY(3-36), Val³hPYY(3-36), Lys²⁵hPYY(3-36), Pro¹³Ala¹⁴hPYY,hPP(1-7)-pNPY, hPP(1-17)-pNPY, Tyr¹NPY, Ala⁷NPY or hPP(19-23)-pNPY.

In another embodiment, the PPF polypeptides of Formula I also do notinclude: Phe²⁷hPYY(3-36), Ile²⁸hPYY(3-36), Val²⁸hPYY(3-36),Gln²⁹hPYY(3-36), Val³⁰hPYY(3-36), Ile³¹hPYY(3-36), Leu³¹hPYY(3-36),Phe³⁶hPYY(3-36), Lys²⁵Phe²⁷hPYY(3-36), Lys²⁵Ile²⁸hPYY(3-36),Lys²⁵Val²⁸hPYY(3-36), Lys²⁵Gln²⁹hPYY(3-36), Lys²⁵Val³⁰hPYY(3-36),Lys²⁵Ile³¹hPYY(3-36), Lys²⁵Leu³¹hPYY(3-36), Lys²⁵Phe³⁶hPYY(3-36),Phe²⁷Ile²⁸hPYY(3-36), Phe²⁷Val²⁸hPYY(3-36), Phe²⁷Gln²⁹hPYY(3-36),Phe²⁷Val³⁰hPYY(3-36), Phe²⁷Ile³¹hPYY(3-36), Phe²⁷Leu³¹hPYY(3-36),Phe²⁷Phe³⁶hPYY(3-36), Gln²⁹Val³⁰hPYY(3-36), Gln²⁹Ile³¹hPYY(3-36),Gln²⁹Leu³¹hPYY(3-36), Gln²⁹Phe³⁶hPYY(3-36), Val³⁰Ile³¹hPYY(3-36),Val³⁰Leu³¹hPYY(3-36), Val³⁰Phe³⁶hPYY(3-36), or Leu³¹Phe³⁶hPYY(3-36).

As will be recognized by one of skill in the art, the polypeptides ofFormula I may be in the free acid form, or may be C-terminally amidated.

1. PYY Analog Polypeptides of the Present Invention

The PYY analog polypeptides of the present invention will generallyinclude at least two PPF motifs including the N-terminal polyproline PPFmotif and the C-terminal tail PPF motif, and will generally retain, atleast in part, a biological activity of native human PYY, e.g., the PYYanalog polypeptides of the present invention will generally be PYYagonists. Moreover, the PYY analog polypeptide will have at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, atleast 94% or at least 97% sequence identity to PYY(3-36). In someembodiments, the PYY analog polypeptides of the present invention willexhibit PYY activity in the treatment and prevention of metabolicconditions and disorders.

In one embodiment, the PYY analog polypeptides of the invention do notinclude any unnatural amino acid resides, and further with the provisiothat the PYY analog polypeptides of the invention do not include anynative PYY polypeptides or 1-4 N-terminal deletions thereof (e.g.,PYY(1-36), PYY(2-36), PYY(3-36)), PYY(4-36)). In some embodiments, thePYY analog polypeptides of the invention do not include: Pro³⁴PYY,His³⁴PYY Lys²⁵hPYY(5-36), Arg⁴hPYY(4-36), Gln⁴hPYY(4-36),Asn⁴hPYY(4-36), Lys²⁵hPYY(4-36), Leu³hPYY(3-36), Val³hPYY(3-36),Lys²⁵hPYY(3-36), Tyr^(1,36)pPYY, Pro¹³Ala¹⁴hPYY, Leu³¹Pro³⁴PYY, FMS-PYY,FMS-PYY(3-36), Fmoc-PYY, Fmoc-PYY(3-36), FMS₂-PYY, FMS₂-PYY(3-36),Fmoc₂-PYY, or Fmoc₂-PYY(3-36).

In another embodiment, such PYY analog polypeptides of the inventionalso do not include: Thr²⁷hPYY(3-36), Ile³⁰hPYY(3-36), Ser³²hPYY(3-36),Lys³³hPYY(3-36), Asn³⁴hPYY(3-36), Lys³⁵hPYY(3-36), Thr³⁶hPYY(3-36),Lys²⁵Thr²⁷hPYY(3-36), Lys²⁵Ile³⁰hPYY(3-36), Lys²⁵Ser³²hPYY(3-36),Lys²⁵Lys³³hPYY(3-36), Lys²⁵Asn²⁴hPYY(3-36), Lys²⁵Lys³⁵hPYY(3-36),Lys²⁵Thr³⁶hPYY(3-36), Thr²⁷Ile²⁸hPYY(3-36), Thr²⁷Val²⁸hPYY(3-36),Thr²⁷Gln²⁹hPYY(3-36), Thr²⁷Ile³⁰hPYY(3-36), Thr²⁷Val³⁰hPYY(3-36),Thr²⁷Ile³¹hPYY(3-36), Thr²⁷Leu³¹hPYY(3-36), Thr²⁷Ser³²hPYY(3-36),Thr²⁷Lys³³hPYY(3-36), Thr²⁷Asn³⁴hPYY(3-36), Thr²⁷Lys³⁵hPYY(3-36),Thr²⁷Thr³⁶hPYY(3-36), Thr²⁷Phe³⁶hPYY(3-36), Phe²⁷Ile³⁰hPYY(3-36),Phe²⁷Ser³²hPYY(3-36), Phe²⁷Lys³³hPYY(3-36), Phe²⁷Asn³⁴hPYY(3-36),Phe²⁷Lys³⁵hPYY(3-36), Phe²⁷Thr³⁶hPYY(3-36), Gln²⁹Ile³⁰hPYY(3-36),Gln²⁹Ser³²hPYY(3-36), Gln²⁹Leu³³hPYY(3-36), Gln²⁹Asn³⁴hPYY(3-36),Gln²⁹Leu³⁵hPYY(3-36), Gln²⁹Thr³⁶hPYY(3-36), Ile³⁰Ile³¹hPYY(3-36),Ile³⁰Leu³¹hPYY(3-36), Ile³⁰Ser³²hPYY(3-36), Ile³⁰Lys³³hPYY(3-36),Ile³⁰Asn³⁴hPYY(3-36), Val³⁰Lys³⁵hPYY(3-36), Ile³⁰Thr³⁶hPYY(3-36),Ile³⁰Phe³⁶hPYY(3-36), Val³⁰Lys³²hPYY(3-36), Val³⁰Lys³³hPYY(3-36),Val³⁰Asn³⁴hPYY(3-36), Val³⁰Lys³⁵hPYY(3-36), Val³⁰Thr³⁶hPYY(3-36),Ile³¹Ser³²hPYY(3-36), Ile³¹Lys³³hPYY(3-36), Ile³¹Asn³⁴hPYY(3-36),Ile³¹Lys³⁵hPYY(3-36), Ile³¹Thr³⁶hPYY(3-36), Ile³¹Phe³⁶hPYY(3-36),Leu³¹Ser³²hPYY(3-36), Leu³¹Lys³³hPYY(3-36), Leu³¹Asn³⁴hPYY(3-36),Leu³¹Lys³⁵hPYY(3-36), Leu³¹Thr³⁶hPYY(3-36), Ser³²Lys³³hPYY(3-36),Ser³²Asn³⁴hPYY(3-36), Ser³²Lys³⁵hPYY(3-36), Ser³²Thr³⁶hPYY(3-36),Ser³²Phe³⁶hPYY(3-36), Lys³³Asn³⁴hPYY(3-36), Lys³³Lys³⁵hPYY(3-36),Lys³³Thr³⁶hPYY(3-36), Lys³³Phe³⁶PYY(3-36), Asn³⁴Lys³⁵hPYY(3-36),Asn³⁴Phe³⁶hPYY(3-36), Lys³⁵Thr³⁶hPYY(3-36), Lys³⁵Phe³⁶hPYY(3-36),Thr²⁷hPYY(4-36), Phe²⁷hPYY(4-36), Ile²⁸hPYY(4-36), Val²⁸hPYY(4-36),Gln²⁹hPYY(4-36), Ile³⁰hPYY(4-36), Val³⁰hPYY(4-36), Ile³¹hPYY(4-36),Leu³¹hPYY(4-36), Ser³²hPYY(4-36), Lys³³PYY(4-36), Asn³⁴hPYY(4-36),Lys³⁵hPYY(4-36), Thr³⁶PYY(4-36), Phe³⁶hPYY(4-36), Lys²⁵Thr²⁷hPYY(4-36),Lys²⁵Phe²⁷hPYY(4-35), Lys²⁵Ile²⁸hPYY(4-36), Lys²⁵Val²⁸hPYY(4-36),Lys²⁵Gln²⁹hPYY(4-36), Lys²⁵Ile³⁰hPYY(4-36), Lys²⁵Val³⁰hPYY(4-36),Lys²⁵Ile³¹hPYY(4-36), Lys²⁵Leu³¹hPYY(4-36), Lys²⁵Ser³²hPYY(4-36),Lys²⁵Lys³³hPYY(4-36), Lys²⁵Asn²⁴hPYY(4-36), Lys²⁵Lys³⁵hPYY(4-36),Lys²⁵Thr³⁶hPYY(4-36), Lys²⁵Phe³⁶hPYY(4-36), Thr²⁷Ile²⁸hPYY(4-36),Thr²⁷Val²⁸hPYY(4-36), Thr²⁷Ile²⁹hPYY(4-36), Thr²⁷Ile³⁰hPYY(4-36),Thr²⁷Val³⁰hPYY(4-36), Thr²⁷Ile³¹hPYY(4-36), Thr²⁷Leu³¹hPYY(4-36),Thr²⁷Ser³²hPYY(4-36), Thr²⁷Lys³³hPYY(4-36), Thr²⁷Asn³⁴hPYY(4-36),Thr²⁷Lys³⁵hPYY(4-36), Thr²⁷Thr³⁶hPYY(4-36), Thr²⁷Phe³⁶hPYY(4-36),Phe²⁷Ile²⁸hPYY(4-36), Phe²⁷Val²⁸hPYY(4-36), Phe²⁷Gln²⁹hPYY(4-36),Phe²⁷Ile³⁰hPYY(4-36), Phe²⁷Val³⁰hPYY(4-36), Phe²⁷Thr³¹hPYY(4-36),Phe²⁷Leu³¹hPYY(4-36), Phe²⁷Ser³²hPYY(4-36), Phe²⁷Lys³³hPYY(4-36),Phe²⁷Asn³⁴hPYY(4-36), Phe²⁷Lys³⁵hPYY(4-36), Phe²⁷Thr³⁶hPYY(4-36),Phe²⁷Phe³⁶hPYY(4-36), Gln²⁹Ile³⁰PYY(4-36), Gln²⁹Val³⁰hPYY(4-36),Gln²⁹Ile³¹hPYY(4-36), Gln²⁹Leu³¹hPYY(4-36), Gln²⁹Ser³²hPYY(4-36),Gln²⁹Leu³³hPYY(4-36), Gln²⁹Asn³⁴hPYY(4-36), Gln²⁹Leu³⁵hPYY(4-36),Gln²⁹Thr³⁶hPYY(4-36), Gln²⁹Phe³⁶hPYY(4-36), Ile³⁰Ile³¹hPYY(4-36),Ile³⁰Leu³¹hPYY(4-36), Ile³⁰Ser³²hPYY(4-36), Ile³⁰Lys³³hPYY(4-36),Ile³⁰Asn³⁴hPYY(4-36), Ile³⁰Lys³⁵hPYY(4-36), Ile³⁰Thr³⁶hPYY(4-36),Ile³⁰Phe³⁶hPYY(4-36), Val³⁰Ile³¹hPYY(4-36), Val³⁰Leu³¹hPYY(4-36),Val³⁰Ser³²hPYY(4-36), Val³⁰Lys³³hPYY(4-36), Val³⁰Asn³⁴hPYY(4-36),Val³⁰Lys³⁵hPYY(4-36), Val³⁰Thr³⁶hPYY(4-36), Val³⁰Phe³⁶hPYY(4-36),Ile³¹Ser³²hPYY(4-36), Ile³¹Lys³³hPYY(4-36), Ile³¹Asn³⁴hPYY(4-36),Ile³¹Lys³⁵hPYY(4-36), Ile³¹Thr³⁶hPYY(4-36), Leu³¹Phe³⁶hPYY(4-36),Leu³¹Phe³⁶hPYY(4-36), Leu³⁴Ser³²hPYY(4-36), Val³¹Lys³³hPYY(4-36),Leu³¹Asn³⁴hPYY(4-36), Leu³¹Lys³⁵hPYY(4-36), Leu³¹Thr³⁶hPYY(4-36),Leu³¹Phe³⁶hPYY(4-36), Ser³²Lys³³hPYY(4-36), Ser³²Asn³⁴hPYY(4-36),Ser³²Lys³⁵hPYY(4-36), Ser³²Thr³⁶hPYY(4-36), Ser³²Phe³⁶hPYY(4-36),Lys³³Asn³⁴hPYY(4-36), Lys³³Lys³⁵hPYY(4-36), Lys³³Thr³⁶hPYY(4-36),Lys³³Phe³⁶hPYY(4-36), Asn³⁴Lys³⁵hPYY(4-36), Asn³⁴Phe³⁶hPYY(4-36),Lys³⁵Thr³⁶PYY(4-36), Lys³⁵Phe³⁶hPYY(4-36), Thr²⁷hPYY(5-36),Phe²⁷hPYY(5-36), Ile²⁸hPYY(5-36), Val²⁸hPYY(5-36), Gln²⁹hPYY(5-36),Ile³⁰hPYY(5-36), Val³⁰hPYY(5-36), Ile³¹hPYY(5-36), Leu³¹hPYY(5-36),Ser³²hPYY(5-36), Lys³³hPYY(5-36), Asn³⁴hPYY(5-36), Lys³⁵hPYY(5-36),Thr³⁶hPYY(5-36), Phe³⁶hPYY(5-36), Lys²⁵Thr²⁷hPYY(5-36),Lys²⁵Phe²⁷hPYY(5-36), Lys²⁵Ile²⁸hPYY(5-36), Lys²⁵Val²⁸hPYY(5-36),Lys²⁵Gln²⁹hPYY(5-36), Lys²⁵Ile³⁰hPYY(5-36), Lys²⁵Val³⁰hPYY(5-36),Lys²⁵Ile³¹hPYY(5-36), Lys²⁵Leu³¹hPYY(5-36), Lys²⁵Ser³²hPYY(5-36),Lys²⁵Lys³³hPYY(5-36), Lys²⁵Asn²⁴hPYY(5-36), Lys²⁵Lys³⁵hPYY(5-36),Lys²⁵Thr³⁶hPYY(5-36), Lys²⁵Phe²⁶hPYY(5-36), Thr²⁷Ile²⁸hPYY(5-36),Thr²⁷Val²⁸hPYY(5-36), Thr²⁷Gln²⁹hPYY(5-36), Thr²⁷Ile³⁰hPYY(5-36),Thr²⁷Val³⁰hPYY(5-36), Thr²⁷Ile³¹hPYY(5-36), Thr²⁷Leu³¹hPYY(5-36),Thr²⁷Ser³²hPYY(5-36), Thr²³Lys³³hPYY(5-36), Thr²⁷Asn³⁴hPYY(5-36),Thr²⁷Lys³⁵hPYY(5-36), Thr²⁷Thr³⁶hPYY(5-36), Thr²⁷Phe³⁶hPYY(5-36),Phe²⁷Ile²⁸hPYY(5-36), Phe²⁷Val²⁸hPYY(5-36), Phe²⁷Gln²⁹hPYY(5-36),Phe²⁷Leu³⁰hPYY(5-36), Phe²⁷Val³⁰hPYY(5-36), Phe²⁷Ile³¹hPYY(5-36),Phe²⁷Leu³¹hPYY(5-36), Phe²⁷Ser³²hPYY(5-36), Phe²⁷Lys³³hPYY(5-36),Phe²⁷Asn³⁴hPYY(5-36), Phe²⁷Lys³⁵hPYY(5-36), Phe²⁷Thr³⁶hPYY(5-36),Phe²⁷Phe³⁶hPYY(5-36), Gln²⁹Ile³⁰hPYY(5-36), Gln²⁹Val³⁰hPYY (5-36),Gln²⁹Ile³¹hPYY(5-36), Gln²⁹Leu³¹hPYY(5-36), Gln²⁹Ser³², hPYY(5-36),Gln²⁹Leu³³hPYY(5-36), Gln²⁹Asn³⁴hPYY(5-36), Gln²⁹Leu³⁵hPYY(5-36),Gln²⁹Thr³⁶hPYY(5-36), Gln²⁹Phe³⁶hPYY(5-36), Ile³⁰Ile³¹hPYY(5-36),Ile³⁰Leu³¹hPYY(5-36), Ile³⁰Ser³²hPYY(5-36), Ile³⁰Lys³³hPYY(5-36),Ile³⁰Asn³⁴hPYY(5-36), Ile³⁰Lys³⁵hPYY(5-36), Ile³⁰Thr³⁶hPYY(5-36),Ile³⁰Phe³⁶hPYY(5-36), Val³⁰Ile³¹hPYY(5-36), Val³⁰Leu³¹hPYY(5-36),Val³⁰Ser³²hPYY(5-36), Val³⁰Lys³³hPYY(5-36), Val³⁰Asn³⁴hPYY(5-36),Val³⁰Lys³⁵hPYY(5-36), Val³⁰Thr³⁶hPYY(5-36), Val³⁰Phe³⁶hPYY(5-36),Ile³¹Ser³²hPYY(5-36), Ile³¹Lys³³hPYY(5-36), Ile³¹Asn³⁴hPYY(5-36),Ile³¹Lys³⁵hPYY(5-36), Ile³¹Thr³⁶hPYY(5-36), Leu³¹Phe³⁶hPYY(5-36),Leu³¹Phe³⁶hPYY(5-36), Leu³¹Ser³²hPYY(5-36), Val³¹Lys³³hPYY(5-36),Leu³¹Asn³⁴hPYY(5-36), Leu³¹Lys³⁵hPYY(5-36), Leu³¹Thr³⁶hPYY(5-36),Leu³¹Phe³⁶hPYY(5-36), Ser³²Lys³³hPYY(5-36), Ser³²Asn³⁴hPYY(5-36),Ser³²Lys³⁵hPYY(5-36), Ser³²Thr³⁶hPYY(5-36), Ser³²Phe³⁶hPYY(5-36),Lys³³Asn³⁴hPYY(5-36), Lys³³Lys³⁵hPYY(5-36), Lys³³Thr³⁶hPYY(5-36),Lys³³Phe³⁶hPYY(5-36), Asn³⁴Lys³⁵hPYY(5-36), Asn³⁴Phe³⁶hPYY(5-36),Lys³⁵Thr³⁶hPYY(5-36), or Lys³⁵Phe³⁶PYY(5-36).

In some embodiments, the PYY analog polypeptides of the invention do notinclude any unnatural amino acid residues, and comprise a C-terminaltail motif of hPYY. The C-terminal motif may comprise amino acidresidues 32-35 of hPYY, e.g., Thr, Arg, Gln, Arg (SEQ ID NO: 351). Insuch an embodiment, the PYY analog polypeptides of the invention do notinclude any native PYY polypeptides or 1-4 N-terminal deletions thereof(e.g., PYY(1-36), PYY(2-36), PYY(3-36) and PYY(4-36)). In someembodiments, such PYY analogs do not include: Lys²⁵hPYY(5-36),Arg⁴hPYY(4-36), Gln⁴hPYY(4-36), Asn⁴hPYY(4-36), Lys²⁵hPYY(4-36),Leu³hPYY(3-36), Val³hPYY(3-36), Lys²⁵hPYY(3-36), Tyr^(1,36)pPYY,Pro¹³Ala¹⁴hPYY, FMS-PYY, FMS-PYY(3-36), Fmoc-PYY, Fmoc-PYY (3-36),FMS₂-PYY, FMS₂-PYY(3-36), Fmoc₂-PYY, or Fmoc₂-PYY(3-36).

In another aspect, such PYY analog polypeptides of the inventioncomprising a C-terminal tail motif of hPYY also do not include:Thr²⁷hPYY(3-36), Ile³⁰hPYY(3-36), Thr³⁶hPYY(3-36), Lys²⁵Thr²⁷hPYY(3-36),Lys²⁵Ile³⁰hPYY(3-36), Lys²⁵Asn²⁴hPYY(3-36), Lys²⁵Thr³⁶hPYY(3-36),Thr²⁷Ile²⁸hPYY(3-36), Thr²⁷Val²⁸hPYY(3-36), Thr²⁷Gln²⁹hPYY(3-36),Thr²⁷Ile³⁰hPYY(3-36), Thr²⁷Val³⁰hPYY(3-36), Thr²⁷Ile³¹hPYY(3-36),Thr²⁷Leu³¹hPYY(3-36), Thr²⁷Thr³⁶hPYY(3-36), Thr²⁷Phe³⁶hPYY(3-36),Phe²⁷Ile³⁰hPYY(3-36), Phe²⁷Thr³⁶PYY(3-36), Gln²⁹Ile³⁰hPYY(3-36),Gln²⁹Thr³⁶hPYY(3-36), Ile³⁰Ile³¹hPYY(3-36), Ile³⁰Leu³¹hPYY(3-36),Ile³⁰Thr³⁶hPYY(3-36), Ile³⁰Phe³⁶PYY(3-36), Val³⁰Thr³⁶hPYY(3-36),Ile³¹Thr³⁶hPYY(3-36), Ile³¹Phe³⁶hPYY(3-36), Leu³¹Thr³⁶hPYY(3-36),Thr²⁷hPYY(4-36), Phe²⁷hPYY(4-36), Ile²⁸hPYY(4-36), Val²⁸hPYY(4-36),Gln²⁹hPYY(4-36), Ile³⁰hPYY(4-36), Val³⁰hPYY(4-36), Ile³¹hPYY(4-36),Leu³¹hPYY(4-36), Thr³⁶hPYY(4-36), Phe³⁶hPYY(4-36), Lys²⁵Thr²⁷hPYY(4-36),Lys²⁵Phe²⁷hPYY(4-36), Lys²⁵Ile²⁸hPYY(4-36), Lys²⁵Val²⁸hPYY(4-36),Lys²⁵Gln²⁹hPYY(4-36), Lys²⁵Ile³⁰hPYY(4-36), Lys²⁵Val³⁰hPYY(4-36),Lys²⁵Ile³¹hPYY(4-36), Lys²⁵Leu³¹hPYY(4-36), Lys²⁵Thr³⁶hPYY(4-36),Lys²⁵Phe³⁶hPYY(4-36), Thr²⁷Ile²⁸hPYY(4-36), Thr²⁷Val²⁸hPYY(4-36),Thr²⁷Gln²⁹hPYY(4-36), Thr²⁷Ile³⁰hPYY(4-36), Thr²⁷Val³⁰hPYY(4-36),Thr²⁷Ile³¹hPYY(4-36), Thr²⁷Leu³¹hPYY(4-36), Thr²⁷Thr³⁶hPYY(4-36),Thr²⁷Phe³⁶hPYY(4-36), Phe²⁷Ile²⁸hPYY(4-36), Phe²⁷Val²⁸hPYY(4-36),Phe²⁷Gln²⁹hPYY(4-36), Phe²⁷Ile³⁰hPYY(4-36), Phe²⁷Val³⁰hPYY(4-36),Phe²⁷Ile³¹hPYY(4-36), Phe²⁷Leu³¹hPYY(4-36), Phe²⁷Thr³⁶hPYY(4-36),Phe²⁷Phe³⁶hPYY(4-36), Gln²⁹Ile³⁰hPYY(4-36), Gln²⁹Val³⁰hPYY(4-36),Gln²⁹Ile³¹hPYY(4-36), Gln²⁹Leu³¹hPYY(4-36), Gln²⁹Thr³⁶hPYY(4-36),Gln²⁹Phe³⁶hPYY(4-36), Ile³⁰Ile³¹hPYY(4-36), Ile³⁰Leu³¹hPYY(4-36),Ile³⁰Thr³⁶hPYY(4-36), Ile³⁰Phe³⁶hPYY(4-36), Val³⁰Ile³¹hPYY(4-36),Val³⁰Leu³¹hPYY(4-36), Val³⁰Thr³⁶hPYY(4-36), Val³⁰Phe³⁶hPYY(4-36),Ile³¹Thr³⁶hPYY(4-36), Leu³¹Phe³⁶hPYY(4-36), Leu³¹Phe³⁶hPYY(4-36),Leu³³Thr³⁶hPYY (4-36), Leu³⁴Phe³⁶hPYY(4-36), Thr²⁷hPYY(5-36),Phe²⁷hPYY(5-36), Ile²⁸hPYY(5-36), Val²⁸hPYY(5-36), Gln²⁹hPYY(5-36),Ile³⁰hPYY(5-36), Val³⁰hPYY(5-36), Ile³¹hPYY(5-36), Leu³¹hPYY(5-36),Thr³⁶hPYY(5-36), Phe³⁶hPYY(5-36), Lys²⁵Thr²⁷hPYY(5-36),Lys²⁵Phe²⁷hPYY(5-36), Lys²⁵Ile²⁸hPYY(5-36), Lys²⁵Val²⁸hPYY(5-36),Lys²⁵Gln²⁹hPYY(5-36), Lys²⁵Ile³⁰hPYY(5-36), Lys²⁵Val³⁰hPYY(5-36),Lys²⁵Ile³¹hPYY(5-36), Lys²⁵Leu³¹hPYY(5-36), Lys²⁵Thr³⁶hPYY(5-36),Lys²⁵Phe³⁶hPYY(5-36), Thr²⁷Ile²⁸hPYY(5-36), Thr²⁷Val²⁸hPYY(5-36),Thr²⁷Gln²⁹hPYY(5-36), Thr²⁷Ile³⁰hPYY(5-36), Thr²⁷Val³⁰hPYY(5-36),Thr²⁷Ile³¹hPYY(5-36), Thr²⁷Leu³¹hPYY(5-36), Thr²⁷Thr³⁶hPYY(5-36),Thr²⁷Phe³⁶hPYY(5-36), Phe²⁷Ile²⁸hPYY(5-36), Phe²⁷Val²⁸hPYY(5-36),Phe²⁷Gln²⁹hPYY(5-36), Phe²⁷Ile³⁰hPYY(5-36), Phe²⁷Val³⁰hPYY(5-36),Phe²⁷Ile³¹hPYY(5-36), Phe²⁷Leu³¹hPYY(5-36), Phe²⁷Thr³⁶hPYY(5-36),Phe²⁷Phe³⁶hPYY(5-36), Gln²⁹Ile³⁰hPYY(5-36), Gln²⁹Val³⁰hPYY(5-36),Gln²⁹Ile³¹hPYY(5-36), Gln²⁹Leu³¹hPYY(5-36), Gln²⁹Thr³⁶hPYY(5-36),Gln²⁹Phe³⁶hPYY(5-36), Ile³⁰Ile³¹hPYY(5-36), Ile³⁰Leu³¹hPYY(5-36),Ile³⁰Thr³⁶hPYY(5-36), Ile³⁰Phe³⁶hPYY(5-36), Val³⁰Ile³¹hPYY(5-36),Val³⁰Leu³¹hPYY(5-36), Val³⁰Thr³⁶hPYY(5-36), Val³⁰Phe³⁶hPYY(5-36),Ile³¹Thr³⁶hPYY(5-36), Leu³¹Phe³⁶PYY(5-36), Leu³¹Phe³⁶PYY(5-36),Leu³¹Thr³⁶hPYY(5-36), or Leu³¹Phe³⁶hPYY(5-36).

In some embodiments, the PYY analog polypeptides of the invention are atleast 34 amino acids in length. In some embodiments, the PYY analogpolypeptides of the invention include only natural L amino acid residuesand/or modified natural L amino acid residues. In some embodiments, thePYY analog polypeptides of the invention do not include unnatural aminoacid residues.

More particularly, in one aspect, the present invention relates to PYYanalog polypeptides including one or more amino acid sequencemodifications. Such modifications include substitutions, insertions,and/or deletions, alone or in combination. In some embodiments, the PYYanalog polypeptides of the invention include one or more modificationsof a “non-essential” amino acid residue. In the context of theinvention, a “non-essential” amino acid residue is a residue that can bealtered, i.e., deleted or substituted, in the native human PYY aminoacid sequence without abolishing or substantially reducing the PYYagonist activity of the PYY analog polypeptide. In some embodiments, thePYY analog polypeptides of the invention retain at least about 25%, orfrom about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, about 95%, about 98%, or about 99% percent of the biologicalactivity of native human PYY with regard to the reduction of nutrientavailability. In another embodiment, the PYY analog polypeptides of theinvention exhibit improved PYY agonist activity. In some embodiments,the PYY analog polypeptides of the invention exhibits at least about110%, about 125%, about 130%, about 140%, about 150%, about 200%, ormore of the biological activity of native human PYY with regard to thereduction of nutrient availability.

PYY analog polypeptides are those having a potency in one of the assaysdescribed herein (including food intake, gastric emptying, pancreaticsecretion, body composition or weight reduction assays) which is equalto or greater than the potency of NPY, PYY, or PYY(3-36) in that sameassay. In some embodiments, PYY analog polypeptides of the invention mayexhibit improved ease of manufacture, stability, and/or ease offormulation, as compared to PP, NPY, PYY, or PYY(3-36).

a. Substitutions

In one embodiment, the PYY analog polypeptides of the invention may haveone or more substitutions in the amino acid sequence of native human PYY(SEQ ID NO: 2), alone or in combination with one or more insertions ordeletions. In some embodiments, the substitution does not abolish orsubstantially reduce the PYY agonist activity of the PYY analogpolypeptide. In one aspect, the present invention relates to PYY analogpolypeptides that have a single substitution, or consecutive ornon-consecutive substitution of more than one amino acid residues in theamino acid sequence of native human PYY (SEQ ID NO: 2). In someembodiments, the PYY analog polypeptides of the invention include one,two, three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions.

In some embodiments, the amino acid residues of native human PYY (SEQ IDNO: 2) at the helical C-terminus region of PYY (e.g., residues 20, 24,25, 27 and 29), the tail end residues (32-36), and/or the N-terminusprolines at position 5 and 8 are not substituted. In some embodiments,amino acid residues are not substituted at positions 32 through 36 ofnative human PYY (SEQ ID NO: 2). In another embodiment, amino acidresidues of native human PYY (SEQ ID NO: 2) are not substituted at oneor more amino acid sequence positions selected from: 5, 7, 8, 20, 24,25, 27, 29, 32, 33, 34, 35, 36, and any combination thereof.

Substitutions may include conserved amino acid substitutions. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain, or physicochemical characteristics (e.g., electrostatic, hydrogenbonding, isosteric, hydrophobic features). Families of amino acidresidues having similar side chains are known in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, methionine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,tryptophan), β-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine).

In another embodiment, the PYY analog polypeptides of the invention mayinclude substitutions of one or more unnatural and/or non-amino acids,e.g., amino acid mimetics, into the sequence of PYY (SEQ ID NO: 2). Insome embodiments, the non-amino acids inserted into the sequence of PYY(SEQ ID NO: 2) may be β-turn mimetics or linker molecules, such as—NH—X—CO—, wherein X=(CH)_(n) (where n can be 2-20) or—NH—CH₂CH₂(—O—CH₂CH₂—O—)_(m)—CH₂—CO— (where m=1-5). Linker molecules caninclude aminocaproyl (“Aca”). β-alanyl, and 8-amino-3,6-dioxaoctanoyl,β-turn mimetics are available commercially (BioQuadrant Inc. Quebec,Canada) and have been described in literature (Hanessian et al.,Tetrahedron 12789-854 (1997); Gu et al., Tetrahedron Letters 44: 5863-6(2003); Bourguet et al., Bioorganic & Medicinal Chemistry Letters 13:1561-4 (2003); Grieco et al., Tetrahedron Letters 43: 6297-9 (2002);Souers et al., Tetrahedron 57: 7431-48 (2001); Tsai et al., Bioorganic &Medicinal Chemistry 7: 29-38 (1999); Virgilio et al., Tetrahedron 53:6635-44 (1997)). β-turn mimetics can include mimic A and mimic Billustrated below.

PYY analog polypeptides comprising amino acid sequence β-turn mimeticsubstitutions include native human PYY (SEQ ID NO: 2), wherein aminoacids at positions x and x+1 are substituted with β-turn mimeticsselected from the group consisting of mimic A and mimic B, wherein x isselected from the amino acids at amino acid positions 8 to 14 of nativehuman PYY. Alternatively, known dipeptide turn inducers may besubstituted, for example, Ala-Aib and Ala-Pro dipeptides.

Other PYY analog polypeptides comprising amino acid sequencesubstitutions include the PYY analog polypeptides of the Formula (II)(SEQ ID NO: 88):

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Pro Xaa₆ Xaa₇ Pro Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈Xaa₁₉ Tyr Xaa₂₁ Xaa₂₂ Xaa₂₃ Leu Arg Xaa₂₆ TyrXaa₂₈ Asn Xaa₃₀ Xaa₃₁ Thr Arg Gln Arg Xaa₃₆wherein:

Xaa₁ is Tyr, Ala, Phe, Trp, or absent;

Xaa₂ is Pro, Gly, d-Ala, homoPro, hydroxy-Pro, or absent;

Xaa₃ is Ile, Ala, NorVal, Val, Leu, Pro, Ser or Thr;

Xaa₄ is Lys, Ala, Gly, Arg, d-Ala, homoLys, homoArg, Glu, or Asp;

Xaa₆ is Glu, Ala, Val, Asp, Asn, or Gln;

Xaa₇ is Ala, Asn, His, Ser, or Tyr;

Xaa₉ is Gly, Ala Ser, sarcosine, Pro, or Aib;

Xaa₁₀ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₁ is Asp, Ala, Glu, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₂ is Ala or d-Ala;

Xaa₁₃ is Ser, Ala, Thr, or homoSer;

Xaa₁₄ is Pro, Ala, homo-Pro, hydroxy-Pro, Aib, or Gly;

Xaa₁₅ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₆ is Glu, Ala, Asp, Asn, or Gln;

Xaa₁₇ is Leu, Ala, Met, Trp, Ile, Val, or NorVal;

Xaa₁₈ is Asn, Asp, Ala, Glu, Gln, Ser or Thr;

Xaa₁₉ is Arg, Tyr, Lys, Ala, Gln, or N(Me)Ala;

Xaa₂₁ is Tyr, Ala, Met, Phe, or Leu;

Xaa₂₂ is Ala, Ser, Thr, or d-Ala;

Xaa₂₃ is Ser, Ala, Thr, or homoSer;

Xaa₂₆ is His or Ala;

Xaa₂₈ is Leu, Ile, Val, or Ala;

Xaa₃₀ is Leu, Ala, NorVal, Val, Ile, or Met;

Xaa₃₁ is Ala, Val, Ile, or Leu; and

Xaa₃₆ is Tyr, N(Me)Tyr, His, Trp, or Phe;

with the proviso that said polypeptide is not a native PPF polypeptide,PYY(2-36), PP(2-36), Ala¹³NPY, Leu³hPYY(3-36), Val³hPYY(3-36),hPP(1-7)-pNPY, or hPP(1-17)-pNPY.

In another embodiment, the PYY analog polypeptides of Formula II also donot include: Ile²⁸hPYY(3-36), Val²⁸hPYY(3-36), Val³⁰hPYY(3-36),Ile³¹hPYY(3-36), Leu³¹hPYY(3-36), Phe³⁶hPYY(3-36), Val³⁰Ile³¹hPYY(3-36),Val³⁰Leu³¹hPYY(3-36), Val³⁰Phe³⁶hPYY(3-36), or Leu³³Phe³⁶hPYY(3-36).

As will be recognized by one of skill in the art, the polypeptides ofFormula II may be in the free acid form, or may be C-terminallyamidated.

Other PYY analog polypeptides comprising amino acid sequencesubstitutions include the PYY analog poly-peptides of the Formula (III)(SEQ ID NO: 348):

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Pro Xaa₆ Xaa₇ Pro Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈Xaa₁₉ Tyr Xaa₂₁ Xaa₂₂ Xaa₂₃ Leu Arg Xaa₂₆ TyrXaa₂₈ Asn Xaa₃₀ Xaa₃₁ Thr Arg Gln Arg Xaa₃₆wherein:

Xaa₁ is Tyr, Phe, Trp, or absent;

Xaa₂ is Pro, Gly, d-Ala, homoPro, hydroxy-Pro, or absent;

Xaa₃ is Ile, Ala, NorVal, Val, Leu, Pro, Ser or Thr;

Xaa₄ is Lys, Ala, Gly, Arg, d-Ala, homoLys, homoArg, Glu, or Asp;

Xaa₆ is Glu, Ala, Val, Asp, Asn, or Gln;

Xaa₇ is Ala, Asn, His, Ser, or Tyr;

Xaa₉ is Gly, Ala Ser, sarcosine, Pro, or Aib;

Xaa₁₀ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₁ is Asp, Ala, Glu, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₂ is Ala or d-Ala;

Xaa₁₃ is Ser, Ala, Thr, Pro, or homoSer;

Xaa₁₄ is Pro, Ala, homo-Pro, hydroxyPro, Aib, or Gly;

Xaa₁₅ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₆ is Glu, Ala, Asp, Asn, or Gly;

Xaa₁₇ is Leu, Ala, Met, Trp, Ile, Val, or NorVal;

Xaa₁₈ is Asn, Asp, Ala, Glu, Gln, Ser or Thr;

Xaa₁₉ is Arg, Tyr, Lys, Ala, Gln, or N(Me)Ala;

Xaa₂₁ is Tyr, Ala, Met, Phe, or Leu;

Xaa₂₂ is Ala, Ser, Thr, or d-Ala;

Xaa₂₃ is Ser, Ala, Thr, or homoSer;

Xaa₂₆ is His or Ala;

Xaa₂₈ is Leu, Ile, Val, or Ala;

Xaa₃₀ is Leu, Ala, NorVal, Val, Ile, or Met;

Xaa₃₁ is Ala, Val, Ile, or Leu; and

Xaa₃₆ is Tyr, N(Me)Tyr, His, Trp, or Phe;

with the proviso that said polypeptide is not a native PPF polypeptide,NPY(2-36), PYY(2-36), PP(2-36), Ala³NPY, Ala⁴NPY, Ala⁶NPY, Ala⁷NPY,Tyr⁷pNPY, Ala⁹NPY, Ala¹⁰NPY, Ala¹¹NPY, Ala¹³NPY, Gly¹⁴NPY, Ala¹⁵NPY,Ala¹⁶NPY, Ala¹⁷NPY, Ala¹⁹NPY, Lys¹⁹NPY, Ala²¹NPY, Ala²²NPY, Lys²⁵NPY,Ala²⁶NPY, Phe²⁷NPY, Ala²⁸NPY, Gln²⁹NPY, Ala³⁰NPY, Ala³¹NPY, Phe³⁶NPY,His³⁶NPY, Leu³hPYY(3-36), Val³hPYY(3-36), Lys²⁵hPYY(3-36),Pro¹³Ala¹⁴hPYY, Tyr¹NPY, Ala⁷NPY, or hPP(19-23)-pNPY.

In another embodiment, the PYY analog polypeptides of Formula III alsodo not include: Ile²⁸hPYY(3-36), Val²⁸hPYY(3-36), Val³⁰hPYY(3-36),Ile³¹hPYY(3-36), Leu³¹hPYY(3-36), Phe³⁶hPYY(3-36), Val³⁰Ile³¹hPYY(3-36),Val³⁰Leu³¹hPYY(3-36), Val³⁰Phe³⁶hPYY(3-36), or Leu³¹Phe³⁶hPYY(3-36).

As will be recognized by one of skill in the art, the polypeptides ofFormula III may be in the free acid form, or may be C-terminallyamidated.

Other PYY analog polypeptides comprising amino acid sequencesubstitutions include the PYY analog polypeptides of the Formula (IV)(SEQ ID NO: 349):

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Pro Xaa₆ Xaa₇ Pro Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈Xaa₁₉ Tyr Xaa₂₁ Xaa₂₂ Xaa₂₃ Leu Arg Xaa₂₆ TyrXaa₂₈ Asn Xaa₃₀ Xaa₃₁ Thr Arg Gln Arg Xaa₃₆wherein:

Xaa₁ is Tyr, Phe, Trp, or absent;

Xaa₂ is Pro, Gly, d-Ala, homoPro, hydroxy-Pro, or absent;

Xaa₃ is Ile, Ala, NorVal, Val, Leu, Pro, Ser or Thr;

Xaa₄ is Lys, Ala, Gly, Arg, d-Ala, homoLys, homoArg, Glu, or Asp;

Xaa₆ is Glu, Ala, Val, Asp, Asn, or Gln;

Xaa₇ is Ala, Asn, His, Ser, or Tyr;

Xaa₉ is Gly, Ala Ser, sarcosine, Pro, or Aib;

Xaa₁₀ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₁ is Asp, Ala, Glu, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₂ is Ala or d-Ala;

Xaa₁₃ is Ser, Ala, Thr, or homoSer;

Xaa₁₄ is Pro, Ala, homo-Pro, hydroxyPro, Aib, or Gly;

Xaa₁₅ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₆ is Glu, Ala, Asp, Asn, or Gln;

Xaa₁₇ is Leu, Ala, Met, Trp, Ile, Val, or NorVal;

Xaa₁₈ is Asn, Asp, Ala, Glu, Gln, Ser or Thr;

Xaa₁₉ is Arg, Tyr, Lys, Ala, Gln, or N(Me)Ala;

Xaa₂₁ is Tyr, Ala, Met, Phe, or Leu;

Xaa₂₂ is Ala, Ser, Thr, or d-Ala;

Xaa₂₃ is Ser, Ala, Thr, or homoSer;

Xaa₂₆ is His or Ala;

Xaa₂₈ is Leu, Ile, Val, or Ala;

Xaa₃₀ is Leu, Ala, NorVal, Val, Ile, or Met;

Xaa₃₁ is Ala, Val, Ile, or Leu; and

Xaa₃₆ is Tyr, N(Me)Tyr, His, Trp, or Phe;

with the proviso that said polypeptide is not a native PPF polypeptide,PYY(2-36), Ala¹³NPY, Leu³hPYY(3-36), or Val³hPYY(3-36).

In another embodiment, the PYY analog polypeptides of Formula IV also donot include: Ile²⁸hPYY(3-36), Val²⁸hPYY(3-36), Val³⁰hPYY(3-36),Ile³¹hPYY(3-36), Leu³¹hPYY(3-36), Phe³⁶hPYY(3-36), Val³⁰Ile³¹hPYY(3-36),Val³⁰Leu³¹hPYY(3-36), Val³⁰Phe³⁶hPYY(3-36), or Leu³¹Phe³⁶hPYY(3-36).

As will be recognized by one of skill in the art, the polypeptides ofFormula IV may be in the free acid form, or may be C-terminallyamidated.

Other PYY analog polypeptides comprising amino acid sequence linkersubstitutions include PYY(1-4)Aminocaproyl(14-36) (IUPAC [Aca⁵⁻¹³]PYY)(Aminocaproyl is abbreviated as “Aca”), PYY(1-4)Aca(15-36),PYY(1-4)Aca(16-36), PYY(1-4)Aca(22-36) (IUPAC [Aca⁵⁻²¹]PYY), andPYY(1-4)Aca(25-36) (IUPAC [Aca⁵⁻²⁴]PYY) (SEQ ID NOS: 180-184).

b. Deletions and Truncations

In another embodiment, the PYY analog polypeptides of the invention mayhave one or more amino acid residues deleted from the amino acidsequence of native human PYY (SEQ ID NO: 2), alone or in combinationwith one or more insertions or substitutions. In one aspect, the PYYanalog polypeptides of the invention may have one or more amino acidresidues deleted from the N-terminus or C-terminus of native human PYY(SEQ ID NO: 2), with the proviso that the polypeptide is not SEQ ID NO:3. In another embodiment, the PYY analog polypeptides of the inventionmay have one or more amino acid residues deleted at amino acid positions2 through 35 of native human PYY (SEQ ID NO: 2). Such deletions mayinclude more than one consecutive or non-consecutive deletions at aminoacid positions 2 through 35 of native human PYY (SEQ ID NO: 2). In someembodiments, the amino acid residues at positions 24 through 36 ofnative human PYY (SEQ ID NO: 2) are not deleted.

In another embodiment, the PPF polypeptides of the invention describedin Formulas I to VII may include N or C-terminal truncations, orinternal deletions at amino acid positions 2 to 35 of Formula I, II,III, IV, V, VI or VII, so long as at least one biological activity of anative PPF polypeptide is retained. In some embodiments, the amino acidresidues at positions 5 through 8 and 24 through 36 are not deleted. Insome embodiments, the amino acid residues at positions 5 through 8 and32 through 35 are not deleted.

c. Insertions

In another embodiment, the PYY analog polypeptides of the invention mayhave one or more amino acid residues inserted into the amino acidsequence of native human PYY (SEQ ID NO: 2), alone or in combinationwith one or more deletions and/or substitutions. In one aspect, thepresent invention relates to PYY analog polypeptides that have a singleinsertion, or consecutive or non-consecutive insertions of more than oneamino acid residues into the amino acid sequence of native human PYY(SEQ ID NO: 2). In some embodiments, amino acid residues are notinserted at positions 24 through 36 of native human PYY (SEQ ID NO: 2).

In another embodiment, the PYY analog polypeptides of the invention mayinclude insertions of one or more unnatural amino acids and/or non-aminoacids into the sequence of PYY (SEQ ID NO: 2). In some embodiments, theunnatural amino acids inserted into the sequence of PYY (SEQ ID NO: 2)may be β-turn mimetics or linker molecules. Linker molecules includeaminocaproyl (“Aca”), β-alanyl, and 8-amino-3,6-dioxaoctanoyl. β-turnmimetics include mimic A and mimic B illustrated below, also Ala-Aib andAla-Pro dipeptides.

In another embodiment, PYY analog polypeptides of the invention mayinclude insertions of polyamino acid sequences (e.g., poly-his,poly-arg, poly-lys, poly-ala, etc.) at either terminus of thepolypeptide, known as “extensions” or “tails.”

PYY analog polypeptides comprising amino acid sequence insertionsinclude alanine substitutions at each amino acid position along thelength of native human PYY. Such PYY analog polypeptides include PYY(+Axa), wherein x is selected from 1′ to 36 (SEQ ID NOS: 54-87).

d. Derivatives

The present invention also relates to derivatives of the PYY analogpolypeptides of the invention. Such derivatives include PYY analogpolypeptides conjugated to one or more water soluble polymer molecules,such as polyethylene glycol (“PEG”) or fatty acid chains of variouslengths (e.g., stearyl, palmitoyl, octanoyl), by the addition ofpolyamino acids, such as poly-his, poly-arg, poly-lys, and poly-ala, orby addition of small molecule substituents include short alkyls andconstrained alkyls (e.g., branched, cyclic, fused, adamantyl), andaromatic groups. In some embodiments, the water soluble polymermolecules will have a molecular weight ranging from about 500 to about20,000 Daltons.

Such polymer-conjugations may occur singularly at the N- or C-terminusor at the side chains of amino acid residues within the sequence of thePYY analog polypeptides. Alternatively, there may be multiple sites ofderivatization along the PYY analog polypeptide. Substitution of one ormore amino acids with lysine, aspartic acid, glutamic acid, or cysteinemay provide additional sites for derivatization. See, e.g., U.S. Pat.Nos. 5,824,784 and 5,824,778. In some embodiments, the PYY analogpolypeptides may be conjugated to one, two, or three polymer molecules.

In some embodiments, the water soluble polymer molecules are linked toan amino, carboxyl, or thiol group, and may be linked by N or C termini,or at the side chains of lysine, aspartic acid, glutamic acid, orcysteine. Alternatively, the water soluble polymer molecules may belinked with diamine and dicarboxylic groups. In some embodiments, thePYY analog polypeptides of the invention are conjugated to one, two, orthree PEG molecules through an epsilon amino group on a lysine aminoacid.

PYY analog polypeptide derivatives of the invention also include PYYanalog polypeptides with chemical alterations to one or more amino acidresidues. Such chemical alterations include amidation, glycosylation,acylation, sulfation, phosphorylation, acetylation, and cyclization. Thechemical alterations may occur singularly at the N- or C-terminus or atthe side chains of amino acid residues within the sequence of the PYYanalog polypeptides. In one embodiment, the C-terminus of these peptidesmay have a free —OH or —NH₂ group. In another embodiment, the N-terminalend may be capped with an isobutyloxycarbonyl group, anisopropyloxycarbonyl group, an n-butyloxycarbonyl group, anethoxycarbonyl group, an isocaproyl group (“isocap”), an octanyl group,an octyl glycine group (denoted as “G(Oct)” or “octylGly”), an8-aminooctanoic acid group, a dansyl, and/or a Fmoc group. In someembodiments, cyclization can be through the formation of disulfidebridges, see. e.g., SEQ ID NO. 171. Alternatively, there may be multiplesites of chemical alteration along the PYY analog polypeptide.

In some embodiments, PYY analog polypeptide derivatives may include PYYanalog polypeptides with chemical alterations to one or more amino acidresidues. These chemical alterations may occur singularly at the N- orC-terminus or at the side chains of amino acid residues within thesequence of the PYY analog polypeptides. In exemplary embodiments, PYYanalog polypeptides are chemically altered to include a Bolton-Huntergroup. Bolton-Hunter reagents are known in the art (“Radioimmunoassayand related methods,” A. E. Bolton and W. M. Hunter, Chapter 26 ofHandbook of Experimental Immunology, Volume I, Immunochemistry, editedby D. M. Weir, Blackwell Scientific Publications, 1986), and may be usedto introduce tyrosine-like moieties with a neutral linkage, throughamino-terminal α-amino groups or ε-amino groups of lysine. In someembodiments, the N-terminal end of a PYY analog polypeptide is modifiedwith a Bolton-Hunter group. In some embodiments, an internal lysineresidue is modified with a Bolton-Hunter group. In some embodiments,there may be multiple sites of Bolton-Hunter modification along the PYYanalog polypeptide. Bolton-Hunter reagents used for polypeptidemodification are commercially available, and may include, but are notlimited to, water-soluble Bolton-Hunter reagent,Sulfosuccinimidyl-3-[4-hydrophenyl]propionate (Pierce Biotechnology,Inc., Rockford, Ill.) and Bolton-Hunter reagent-2, N-Succinimidyl3-(4-hydroxy-3-iodophenyl) Propionate (Wako Pure Chemical Industries,Ltd., Japan, catalog #199-09341). An exemplary Bolton-Hunter groupconjugated through an amide linkage to a PYY analog polypeptide isillustrated below, wherein the dashed line passes through the amidebond:

PYY analog polypeptides may be iodinated (such as radiolabeled with¹²⁵I) before or after Bolton-Hunter modification. ¹²⁵I-Bolton-Hunterlabeled PYY or PYY analogs may also be purchased from AmershamCorporation (Arlington Heights, Ill.). Bolton-Hunter derivatives areabbreviated as “BH-modified” in Table 4. (SEQ ID NOS: 475-480).

e. Analogs and Derivatives

In some embodiments, the PYY analog polypeptides include combinations ofthe above-described modifications, i.e., deletion, insertion, andsubstitution.

By way of example, PYY analog polypeptides may include N-terminaldeletions in combination with one or more amino acid substitutions. Forinstance, PYY analog polypeptides include PYY (3-36) with the one ormore of the following amino acid substitutions: Ala³, Leu³, Pro³, Ala⁴,Gly⁴, d-Ala⁴, homoLys⁴, Glu⁴, Ala⁵, Ala⁶, Val⁶, d-Ala⁷, Tyr⁷, His⁷,Ala⁸, Ala⁹, Ala¹⁰, Ala¹¹, d-Ala¹², Ala¹³, homoSer¹³, Ala¹⁴, Ala¹⁵,Gln¹⁵, Ala¹⁶, Ala¹⁷, Met¹⁷, Ala¹⁸, Ser¹⁸, nor-Val¹⁸, Ala¹⁹, N-Me-Ala¹⁹,Lys¹⁹, homoArg¹⁹, Ala²⁰, Ala²¹, d-Ala²², Ala²³, Ala²⁴, Ala²⁵, Lys²⁵,homoArg²⁵, Ala²⁶, Ala²⁷, Ala²⁸, Ala²⁹, Ala³⁰, Ala³¹, Ala³², Ala³³,Lys³³, Ala³⁴, Ala³⁵, Ala³⁶, His³⁶, Trp³⁶, N-Me-Tyr³⁶, and Phe³⁶. In someembodiments, the PYY analog polypeptide includes one, two, or threeamino acid substitutions. Certain PYY analog polypeptides comprisedeletions in combination with amino acid insertions. (see, e.g., SEQ IDNOS: 89-174)

PYY analog polypeptides include the polypeptides of the Formula (V) (SEQID NO: 350):

Xaa₃ Xaa₄ Pro Xaa₆ Xaa₇ Pro Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ TyrXaa₂₁ Xaa₂₂ Xaa₂₃ Leu Arg Xaa₂₆ Tyr Xaa₂₈ AsnXaa₃₀ Xaa₃₁ Thr Arg Gln Arg Xaa₃₆wherein:

Xaa₃ is Ile, Ala, Pro, Ser, Thr, or NorVal;

Xaa₄ is Lys, Ala, Gly, Glu, Asp, d-Ala, homoLys, or homoArg;

Xaa₆ is Glu, Ala, Vat, Asp, Asn, or Gln;

Xaa₇ is Ala, Asn, His, Set, or Tyr;

Xaa₉ is Gly, Ala, Ser, sarcosine, Pro, or Aib;

Xaa₁₀ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₁ is Asp, Ala, Glu, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₂ is Ala or d-Ala;

Xaa₁₃ is Ser, Ala, Thr, or homoSer;

Xaa₁₄ is Pro, Ala, homoPro, hydroxyPro, Aib, or Gly;

Xaa₁₅ is Glu, Ala, Asp, Asn, Gln, Pro, Aib, or Gly;

Xaa₁₆ is Glu, Ala, Asp, Asn, or Gln;

Xaa₁₇ is Leu, Ala, Met, Trp, Ile, Val, or NorVal;

Xaa₁₈ is Asn, Asp, Ala, Glu, Gln, Ser or Thr;

Xaa₁₉ is Arg, Tyr, Lys, Ala, Gln, or N(Me)Ala;

Xaa₂₁ is Tyr, Ala, Met, Phe, or Leu;

Xaa₂₂ is Ala, Ser, Thr, or d-Ala;

Xaa₂₃ is Ser, Ala, Thr, or homoSer;

Xaa₂₆ is His or Ala;

Xaa₂₈ is Leu or Ala;

Xaa₃₀ is Leu, Ala, NorVal, or Ile;

Xaa₃₁ is Ala or Val; and

Xaa₃₆ is Tyr, N(Me)Tyr, His, or Trp;

with the proviso that said polypeptide is not a native PPF polypeptide.

As will be recognized by one of skill in the art, the polypeptides ofFormula V may be in the free acid form, or may be C-terminally amidated.

Also included within the scope of the invention are PYY analogpolypeptides of Formulas II to VII, wherein the indicated amino acidresidue is chemical modified or derivatized (e.g., through fatty acidderivitization, PEGylation, amidation, glycolization, etc.). Alsocontemplated within the scope of the invention are D-amino acid residuesof the indicated amino acids.

In some embodiments, PYY analog polypeptides include the polypeptides ofFormulas II to VII with internal deletions, particularly in areas notcorresponding to the C-terminal tail PPF motif, as described herein.

PYY analog polypeptides comprising substitutions of unnatural aminoacids include PYY(3-36), wherein amino acids at positions x and x+1 aresubstituted with β-turn mimetics selected from the group consisting ofmimic A and mimic B, wherein x is selected from positions 8 to 14 (see,e.g., SEQ ID NOS: 211-217 and 231-237).

Derivatives of the PYY analog polypeptides of the invention can includepolymer-conjugated PYY analog polypeptides, wherein the PYY analogpolypeptide includes any of the above-described insertions, deletions,substitutions, or combinations thereof, and the polymer molecule isconjugated at a lysine residue. Other derivatives of PYY analogpolypeptides include PYY, PYY(3-36) or PYY(4-36) with the followingsubstitutions and alterations: [Lys⁴-fatty acid chain]PYY(3-36);[Lys⁴-fatty acid chain]PYY(4-36); [Ala²Lys¹⁹-fatty acid chain]PYY(3-36);[Ile³-fatty acid chain]PYY(3-36); [Ser¹³-OAc]PYY(3-36) (OAc isO-Acylation with fatty acids or acetyl groups); [Ser²³-OAc]PYY(3-36):[Ile²-Octanoyl chain]PYY(3-36); [Lys¹⁹-Octanoyl chain]PYY(3-36); and[Lys¹⁹-Stearyl chain]PYY(3-36). (see e.g., SEQ ID NOS: 185-208).

Further examples of the PYY analog polypeptides of the present inventionare provided in the Sequence Listing and discussed in the Examplessection below.

2. PPF Chimeric Polypeptides

In yet another aspect of the invention, the PPF polypeptides of theinvention include PPF chimeric polypeptides comprising a fragment of aPP, PYY or NPY polypeptide covalently linked to at least one additionalfragment of a second PP, PYY or NPY polypeptide, wherein each PP, PYY orNPY fragment includes a PPF motif. Alternatively, the PPF chimericpolypeptides of the invention may comprise a fragment of a PP familypolypeptide linked to one, two, three, or four polypeptides segments,wherein at least one of the linked polypeptide segments is a fragment ofa second PP family polypeptide. In certain embodiments, PPF polypeptidesdo not include an N-terminal PP fragment with a C-terminal NPY fragment.PPF chimeric polypeptides of the invention will exhibit at least 50%sequence identity to a native PYY(3-36) over the entire length of thePYY(3-36). In some embodiments, such PPF chimeric polypeptides of theinvention may exhibit at least 60%, at least 70%, at least 80%, at least90%, at least 92%, at least 94% or at least 97% sequence identity to anative PYY(3-36) over the entire length of the PYY(3-36). Such PPFchimeric polypeptides of the invention may also exhibit at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 92%, atleast 94% or at least 97% sequence identity to a native PP. In yetanother embodiment, such PPF chimeric polypeptides of the invention mayexhibit at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 92%, at least 94% or at least 97% sequence identity to anative NPY. In some embodiments, the PPF chimeric polypeptides of theinvention include at least the N-terminal polyproline PPF motif and theC-terminal tail PPF motif.

Again, the PPF polypeptides of the present invention will generallyretain, at least in part, a biological activity of native human PP, PYY,or NPY. In some embodiments, the PPF chimeric polypeptides of thepresent invention will exhibit biological activity in the treatment andprevention of metabolic conditions and disorders.

The polypeptide fragments may be covalently linked together in anymanner known in the art, including but not limited to direct amide bondsor chemical linker groups. Chemical linker groups may include peptidemimetics which induce or stabilize polypeptide conformation. PPFchimeric polypeptides of the invention include PYY-PP, PYY-NPY, PP-PYY,PP-NPY, NPY-PP, or NPY-PYY chimeras.

The PPF chimeric polypeptides of the invention may be at least 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 amino acids in length.In some embodiments, the PYY analog polypeptides of the inventioninclude only natural L amino acid residues and/or modified natural Lamino acid residues. In some embodiments, the PYY analog polypeptides ofthe invention do not include unnatural amino acid residues.

In some embodiments, the PPF chimeric polypeptides of the invention donot include: hPP(1-7)-pNPY, hPP(1-17)-pNPY, hPP(19-23)-pNPY,hPP(19-23)-Pro³⁴pNPY, hPP(19-23)-His³⁴pNPY, rPP(19-23)-pNPY,rPP(19-23)-Pro³⁴pNPY, rPP(19-23)-His³⁴pNPY, hPP(1-17)-His³⁴pNPY,pNPY(1-7)-hPP, pNPY(1-7, 19-23)-hPP, cPP(1-7)-pNPY(19-23)-hPP,cPP(1-7)-NPY(19-23)-His³⁴hPP, hPP(1-17)-His³⁴pNPY, hPP(19-23)-pNPY,hPP(19-23)-Pro³⁴pNPY, pNPY(1-7)-hPP, pNPY(19-23)-hPP,pNPY(19-23)-Gln³⁴hPP, pNPY(19-23)-His³⁴hPP, pNPY(19-23)-Phe⁶Gln³⁴hPP,pNPY(19-23)-Phe⁶His³⁴hPP, pNPY(1-7, 19-23)-hPP, pNPY(1-7,19-23)-Gln³⁴hPP, cPP(20-23)-Pro³⁴-pNPY, cPP(21-23)-Pro³⁴-pNPY,cPP(22-23)-Pro³⁴-pNPY, cPP(1-7)-Pro³⁴-pNPY, cPP(20-23)-Pro³⁴-pNPY,cPP(1-7,20-23)-Pro³⁴-pNPY, cPP(1-7)-pNPY(19-23)-hPP,cPP(1-7)-pNPY(19-23)-His³⁴hPP, cPP(1-7)-gPP(19-23)-hPP,cPP(1-7)-pNPY(19-23)-Ala³¹Aib³²Gln³⁴-hPP,cPP(1-7)-pNPY(19-23)-Ala³¹Aib³²His³⁴-hPP hPP(1-7)-Ala³¹Aib³²-pNPY,hPP(1-17)-Ala³¹Aib³²-pNPY, pNPY(1-7)-Ala³¹Aib³²Gln³⁴-hPP, or pNPY(1-7,19-23)-Ala³¹Aib³²Gln³⁴-hPP.

In some embodiments, the PPF chimeric polypeptides of the invention maycomprise fragments of PP family analog polypeptides. For instance, thePPF chimeric polypeptides may comprise PPF analog polypeptides describedherein, as well as PP analog polypeptides, and NPY analog polypeptides.

PYY analog polypeptide are those having a potency in one of the assaysdescribed herein (including food intake, gastric emptying, pancreaticsecretion, body composition or weight reduction assays) which is equalto or greater than the potency of NPY, PYY, or PYY(3-36) in that sameassay. In some embodiments, PYY analog polypeptides of the invention mayexhibit improved ease of manufacture, stability, and/or ease offormulation, as compared to PP, NPY, PYY, or PYY(3-36).

In some embodiments, the PPF chimeric polypeptides of the inventionretain at least about 25%, or from about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, orabout 99% percent of the biological activity of native human PYY withregard to the reduction of nutrient availability, the reduction of foodintake, the effect of body weight gain, and/or the treatment andprevention of metabolic conditions and disorders. In another embodiment,the PPF chimeric polypeptides of the invention exhibit improved PYYagonist activity. In some embodiments, the PPF chimeric polypeptides ofthe invention exhibits at least about 110%, about 125%, about 130%,about 140%, about 150%, about 200%, or more of the biological activityof native human PYY with regard to the reduction of nutrientavailability the reduction of food intake, the effect of body weightgain, and/or the treatment and prevention of metabolic conditions anddisorders.

More particularly, in one aspect, the PPF chimeric polypeptides comprisea fragment of PP linked to a fragment of PYY. In one embodiment, the PPFchimeric polypeptides of the invention comprise an N-terminal fragmentof PP or a PP analog polypeptide linked at its C-terminal end to aC-terminal fragment of PYY or a PYY analog polypeptide. In anotherembodiment, the PPF chimeric polypeptides of the invention comprise anN-terminal fragment of PYY, PYY(3-36), or a PYY analog polypeptidelinked at its C-terminal end to a C-terminal fragment of PP or a PPanalog polypeptide.

In some embodiments, the PPF chimeric polypeptides comprise a fragmentof PYY linked to a fragment of NPY. In one embodiment, the PPF chimericpolypeptides of the invention comprise an N-terminal fragment of PYY,PYY(3-36), or a PYY analog polypeptide linked at its C-terminal end to aC-terminal fragment of NPY or a NPY analog polypeptide. In anotherembodiment, the PPF chimeric polypeptides of the invention comprise anN-terminal fragment of NPY or a NPY analog polypeptide linked at itsC-terminal end to a C-terminal fragment of PYY or a PYY analogpolypeptide.

In some embodiments, the PPF chimeric polypeptides comprise a fragmentof PP linked to a fragment of NPY. In one embodiment, the PPF chimericpolypeptides of the invention comprise an N-terminal fragment of PP or aPP analog polypeptide linked at its C-terminal end to a C-terminalfragment of NPY or a NPY analog polypeptide. In another embodiment, thePPF chimeric polypeptides of the invention comprise an N-terminalfragment of NPY or a NPY analog polypeptide linked at its C-terminal endto a C-terminal fragment of PP or a PP analog polypeptide.

In some embodiments, a fragment of PP, a PP analog polypeptide, PYY,PYY(3-36), a PYY analog polypeptide, NPY, or an NPY analog polypeptideis a fragment comprising anywhere from 4 to 20 amino acid residues ofthe PP, PP analog polypeptide, PYY, PYY(3-36), PYY analog polypeptide,NPY, or NPY analog polypeptide. In some embodiments, the length offragment is selected so as to obtain a final PPF chimeric polypeptide ofat least 34 amino acids in length.

The PPF chimeric polypeptides of the present invention may also comprisefurther modifications including, but are not limited to, substitution,deletion, and insertion to the amino acid sequence of such PPF chimericpolypeptides and any combination thereof. In some embodiments, the PPFchimeric polypeptides of the invention include one or more modificationsof a “non-essential” amino acid residue. In the context of theinvention, a “non-essential” amino acid residue is a residue that can bealtered, i.e., deleted or substituted, in the native human amino acidsequence of the fragment. e.g., the PP family polypeptide fragment,without abolishing or substantially reducing the PYY agonist activity ofthe PPF chimeric polypeptide.

The present invention also relates to derivatives of the PPF chimericpolypeptides. Such derivatives include PPF chimeric polypeptidesconjugated to one or more water soluble polymer molecules, such aspolyethylene glycol (“PEG”) or fatty acid chains of various lengths(e.g., stearyl, palmitoyl, octanoyl, alkyl etc.), or by the addition ofpolyamino acids, such as poly-his, poly-arg, poly-lys, and poly-ala.Modifications to the PPF chimeric polypeptides can also include smallmolecule substituents, such as short alkyls and constrained alkyls(e.g., branched, cyclic, fused, adamantyl), and aromatic groups. In someembodiments, the water soluble polymer molecules will have a molecularweight ranging from about 500 to about 20,000 Daltons.

Such polymer-conjugations and small molecule substituent modificationsmay occur singularly at the N- or C-terminus or at the side chains ofamino acid residues within the sequence of the PPF chimericpolypeptides. Alternatively, there may be multiple sites ofderivatization along the PPF chimeric polypeptide. Substitution of oneor more amino acids with lysine, aspartic acid, glutamic acid, orcysteine may provide additional sites for derivatization. See, e.g.,U.S. Pat. Nos. 5,824,784 and 5,824,778. In some embodiments, the PPFchimeric polypeptides may be conjugated to one, two, or three polymermolecules.

In some embodiments, the water soluble polymer molecules are lined to anamino, carboxyl, or thiol group, and may be linked by N or C terminus,or at the side chains of lysine, aspartic acid, glutamic acid, orcysteine. Alternatively, the water soluble polymer molecules may belinked with diamine and dicarboxylic groups. In some embodiments, thePPF chimeric polypeptides of the invention are conjugated to one, two,or three PEG molecules through an epsilon amino group on a lysine aminoacid.

PPF chimeric polypeptide derivatives of the invention also include PPFchimeric polypeptides with chemical alterations to one or more aminoacid residues. Such chemical alterations include amidation,glycosylation, acylation, sulfation, phosphorylation, acetylation, andcyclization. The chemical alterations may occur singularly at the N- orC-terminus or at the side chains of amino acid residues within thesequence of the PPF chimeric polypeptides. In one embodiment, theC-terminus of these peptides may have a free —OH or —NH₂ group. Inanother embodiment, the N-terminal end may be capped with anisobutyloxycarbonyl group, an isopropyloxycarbonyl group, ann-butyloxycarbonyl group, an ethoxycarbonyl group, an isocaproyl group(isocap), an octanyl group, an octyl glycine group (G(Oct)), or an8-aminooctanoic acid group. In some embodiments, cyclization can bethrough the formation of disulfide bridges. Alternatively, there may bemultiple sites of chemical alteration along the PYY analog polypeptide.

In some embodiments, the PPF chimeric polypeptides include those havingan amino acid sequence of SEQ ID NOs. 238-347.

Examples of the PPF chimeric polypeptides of the present invention areprovided in the Sequence Listing and further discussed in the Examplessection below.

Other PPF polypeptides include polypeptides of the Formula (VI) (SEQ IDNO: 481):

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Pro Glu Xaa₇ Pro Xaa₉ GluAsp Xaa₁₂ Xaa₁₃ Xaa₁₄ Glu Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉Try Xaa₂₁ Xaa₂₂ Xaa₂₃ Leu Xaa₂₅ Xaa₂₆ Tyr Xaa₂₈Asn Xaa₃₀ Xaa₃₁ Thr Arg Gln Xaa₃₅ Xaa₃₆wherein:

Xaa₁ is Tyr or absent;

Xaa₂ is Ile, Pro, or absent;

Xaa₃ is Ile, BH-modified Lys, Lys, Val, or Pro;

Xaa₄ is Lys, BH-modified Lys, Ala, Ser, or Arg;

Xaa₇ is Ala, Gly, or His;

Xaa₉ is Gly or Ala;

Xaa₁₂ is Ala or Pro;

Xaa₁₃ is Ser or Pro;

Xaa₁₄ is Pro, Ala, or Ser;

Xaa₁₆ is Glu or Asp;

Xaa₁₇ is Leu or Ile;

Xaa₁₈ is Asn or Ala;

Xaa₁₉ is Arg, Lys, BH-modified Lys, Gln, or N(Me)Ala;

Xaa₂₁ is Tyr, Ala, Phe, Lys or BH-modified Lys;

Xaa₂₂ is Ala or Ser;

Xaa₂₃ is Ser, Ala, or Asp;

Xaa₂₅ is Arg, Lys or BH-modified Lys;

Xaa₂₆ is His, Ala, or Arg;

Xaa₂₈ is Leu or Ile;

Xaa₃₀ is Leu or Met;

Xaa₃₁ is Val, Ile, or Leu;

Xaa₃₅ is Lys, BH-modified Lys, or Arg; and

Xaa₃₆ is Tyr, Trp, or Phe;

with the proviso that said PPF polypeptide is not a native PPFpolypeptide, PYY(2-36), Val³hPYY(3-36), Lys²³PYY(3-36),Lys²⁵Ile²⁸hPYY(3-36), Lys²³Ile³¹hPYY(3-36), Lys²⁵Leu³³hPYY(3-36),Lys²⁵Phe³⁶hPYY(3-36), Ile²⁸hPYY(3-36), Ile³¹hPYY(3-36), Leu³¹hPYY(3-36),Phe³⁶hPYY(3-36), Leu³¹Phe³⁶hPYY(3-36), or Pro¹³Ala¹⁴hPYY.

As will be recognized by one of skill in the art, the polypeptides ofFormula VI may be in the free acid form, or may be C-terminallyamidated.

In some embodiments, the PPF polypeptide may comprise an N-terminalfragment consisting essentially of the first 17 amino acid residues ofnative human PYY (SEQ ID NO: 2) linked to a C-terminal fragmentconsisting essentially of amino acid residues 18-36 of native human NPY(SEQ ID NO: 4), wherein one or more amino acid residues at theN-terminus of the PYY fragment may be deleted or absent, and whereinone, two, three, four, five, six, seven, eight, nine or ten amino acidsubstitutions may be made in each of the PYY and NPY fragments. In someembodiments, an N-terminal fragment consisting essentially of the first17 amino acids of the PPF polypeptide may exhibit at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 92%, at least94% or at least 97% sequence identity to the first 17 amino acids of anative PYY. In some embodiments, a C-terminal fragment of the PPFpolypeptide consisting essentially of amino acid residues 18-36 mayexhibit at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 92%, at least 94% or at least 97% sequence identity toamino acids 18-36 of a native NPY. In some embodiments, amino acids inthe N-terminal fragment of PYY (e.g., prolines at position 5 and 8,glutamates at positions 6, 10 and 15, or aspartate at position 11),and/or amino acids in the C-terminal fragment of NPY (e.g., tyrosines atpositions 20 and 27, leucine at position 24, asparagine at position 29,threonine at position 32, arginine at position 33, or glutamine atposition 34) are not substituted. In some embodiments, the PPFpolypeptides include those having an amino acid sequence of SEQ ID Nos.266, 267, 274, 282, 320, and 436 to 480. In some embodiments, the PPFpolypeptides further comprise an N-terminal cap. Examples of these PPFpolypeptides include SEQ ID NOs: 282, 320, 437, 441, 444, 445-447, 452,454-459, 461-464, 466, 468-470 and 472-480.

Other PPF polypeptides include polypeptides of the Formula (VII) (SEQ IDNO: 482):

Xaa₁ Xaa₂ Pro Xaa₄ Pro Xaa₆ His Pro Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ AlaXaa₁₉ Tyr Xaa₂₁ Xaa₂₂ Xaa₂₃ Leu Xaa₂₅ Xaa₂₆ Xaa₂₇Xaa₂₈ Xaa₂₉ Xaa₃₀ Xaa₃₁ Thr Arg Gln Arg Tyrwherein:

Xaa₁ is Tyr or absent;

Xaa₂ is Ile, Pro, or absent;

Xaa₄ is Lys, BH-modified Lys, Ala, Ser, or Arg;

Xaa₆ is Glu, Gln, Ala, Asn, Asp, or Val;

Xaa₉ is Gly or Ala;

Xaa₁₀ is Glu, Ala, Asp, Asn, Gln, Gly, Pro, or Aib;

Xaa₁₁ is Glu, Ala, Asp, Asn, Gln, Gly, Pro, or Aib;

Xaa₁₂ is Ala or Pro;

Xaa₁₃ is Ser or Pro;

Xaa₁₄ is Pro, Ala, or Ser;

Xaa₁₅ is Glu, Ala, Asp, Asn, Gln, Gly, Pro, or Aib;

Xaa₁₆ is Glu or Asp;

Xaa₁₇ is Leu or Ile;

Xaa₁₉ is Arg, Lys, BH-modified Lys, Gln, or N(Me)Ala;

Xaa₂₁ is Tyr, Ala, Phe, Lys, or BH-modified Lys;

Xaa₂₂ is Ala or Ser;

Xaa₂₃ is Ser, Ala, or Asp;

Xaa₂₅ is Arg, Lys or BH-modified Lys;

Xaa₂₆ is His, Ala, or Arg;

Xaa₂₇ is Tyr, or Phe;

Xaa₂₈ is Leu or Ile;

Xaa₂₉ is Asn, or Gln;

Xaa₃₀ is Leu or Met; and

Xaa₃₁ is Val, Ile, or Leu.

As will be recognized by one of skill in the art, the polypeptides ofFormula VII may be in the free acid form, or may be C-terminallyamidated.

In some embodiments, the PPF polypeptide may comprise an N-terminalfragment consisting essentially of the first 17 amino acid residues ofnative human PYY (SEQ ID NO: 2) linked to a C-terminal fragmentconsisting essentially of amino acid residues 18-36 of native human NPY(SEQ ID NO: 4), wherein one or more amino acid residues at theN-terminus of the PYY fragment may be deleted or absent, and whereinone, two, three, four, five, six, seven, eight, nine or ten amino acidsubstitutions may be made in each of the PYY and NPY fragments. In someembodiments, an N-terminal fragment consisting essentially of the first17 amino acids of the PPF polypeptide may exhibit at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 92%, at least94% or at least 97% sequence identity to the first 17 amino acids of anative PYY. In some embodiments, a C-terminal fragment of the PPFpolypeptide consisting essentially of amino acid residues 18-36 mayexhibit at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 92%, at least 94% or at least 97% sequence identity toamino acids 18-36 of a native NPY. In some embodiments, amino acids inthe N-terminal fragment of PYY (e.g., prolines at positions 3, 5 and 8,or histidine 7), and/or amino acids in the C-terminal fragment of NPY(e.g., alanine at position 18, tyrosines at positions 20 and 36, leucineat position 24, threonine at position 32, arginine at position 33,glutamine at position 34, or arginine at position 35) are notsubstituted. In some embodiments, the PPF polypeptides include thosehaving an amino acid sequence of SEQ ID Nos. 266, 437, 438, 439, 442,462, 469, 470, 471 and 472. In some embodiments, the PPF polypeptidesfurther comprise an N-terminal cap. Examples of these PPF polypeptidesinclude SEQ ID NOs: 437, 462, 469, 470 and 472.

Examples of the PPF polypeptides of the present invention are providedin the Sequence Listing and further discussed in the Examples sectionbelow.

Use of PPF Polypeptides in the Treatment or Prevention of MetabolicConditions or Disorders

It has been generally accepted that endogenous NPY (reviewed in Schwartzet al., Nature 404: 661-71 (2000)) and PYY (Morley et al., Brain Res.341: 200-3 (1985)), via their receptors, increase feeding behavior.Methods directed at therapies for obesity have invariably attempted toantagonize Y receptors, while claims for treating anorexia have beendirected at agonists of this ligand family. However, as described andclaimed in the commonly-owned pending U.S. Patent Application No.20020141985, it has been surprisingly discovered that peripheraladministration of PYY analog polypeptides has a potent effect to reducenutrient availability (see also Batterham et al., Nature 418: 650-4,2002; WO 03/026591; and WO 03/057235), rather than increase it assuggested by reports in the patent and scientific literature (see, e.g.,U.S. Pat. Nos. 5,912,227 and 6,315,203 which disclose the use of PYYreceptor agonists to increase weight gain). The spectrum of actions ofinhibition of food intake, slowing of gastric emptying, inhibition ofgastric acid secretion, and inhibition of pancreatic enzyme secretion,are useful to exert clinical benefit in metabolic diseases such as type1, type 2, or gestational diabetes mellitus, obesity and othermanifestations of insulin-resistance syndrome (Syndrome X), and in anyother use for reducing nutrient availability.

As such, in another aspect of the invention, methods for treating orpreventing obesity are provided, wherein the method comprisesadministering a therapeutically or prophylactically effective amount ofa PPF polypeptide to a subject in need thereof. In some embodiments, thesubject is an obese or overweight subject. While “obesity” is generallydefined as a body mass index over 30, for purposes of this disclosure,any subject, including those with a body mass index of less than 30, whoneeds or wishes to reduce body weight is included in the scope of“obese.” Subjects who are insulin resistant, glucose intolerant, or haveany form of diabetes mellitus (e.g., type 1, 2 or gestational diabetes)can benefit from this method.

In other aspects of the invention, methods of reducing food intake,reducing nutrient availability, causing weight loss, affecting bodycomposition, and altering body energy content or increasing energyexpenditure, treating diabetes mellitus, and improving lipid profile(including reducing LDL cholesterol and/or triglyceride levels and/orchanging HDL cholesterol levels) are provided, wherein the methodscomprise administering to a subject an effective amount of a PPFpolypeptide of the invention. In some embodiments, the methods of theinvention are used to treat or prevent conditions or disorders which canbe alleviated by reducing nutrient availability in a subject in needthereof, comprising administering to said subject a therapeutically orprophylactically effective amount of a PPF polypeptide of the invention.Such conditions and disorders include, but are not limited to,hypertension, dyslipidemia, cardiovascular disease, eating disorders,insulin-resistance, obesity, and diabetes mellitus of any kind.

Without intending to be limited by theory, it is believed that theeffects of peripherally-administered PPF polypeptides of the presentinvention in the reduction of food intake, in the delay of gastricemptying, in the reduction of nutrient availability, and in thecausation of weight loss are determined by interactions with one or moreunique receptor classes in, or similar to, those in the PP family. Moreparticularly, it appears that a receptor or receptors similar to thePYY-preferring (or Y7) receptors are involved.

Additional assays useful to the invention include those that candetermine the effect of PPF compounds on body composition. An exemplaryassay can be one that involves utilization of a diet-induced obese (DIO)mouse model for metabolic disease. Prior to the treatment period, maleC57BL/6J mice can be fed a high-fat diet (#D12331, 58% of calories fromfat; Research Diets, Inc.) for 6 weeks beginning at 4 weeks of age.During the study, the mice can continue to eat their high-fat diet.Water can be provided ad libitum throughout the study. One group ofsimilarly-aged non-obese mice can be fed a low-fat diet (#112329, 11% ofcalories from fat) for purposes of comparing metabolic parameters to DIOgroups.

DIO mice can be implanted with subcutaneous (SC) intrascapular osmoticpumps to deliver either vehicle (50% dimethylsulfoxide [DMSO] in water)n=20 or a compound of the invention n=12. The pumps of the latter groupcan be set to deliver any amount, e.g., 1000 μg/kg/d of a compound ofthe invention for 7 days.

Body weights and food intake can be measured over regular intervalsthroughout the study periods. Respiratory quotient (RQ, defined as CO₂production÷O₂ consumption) and metabolic rate can be determined usingwhole-animal indirect calorimetry (Oxymax, Columbus Instruments,Columbus, Ohio). The mice can be euthanized by isoflurane overdose, andan index of adiposity (bilateral epididymal fat pad weight) measured.Moreover, prior to determination of epididymal weight, body composition(lean mass, fat mass) for each mouse can be analyzed using a Dual EnergyX-ray Absorptiometry (DEXA) instrument per manufacturer's instructions(Lunar Piximus, GE Imaging System). In some embodiments, PPFpolypeptides of the invention are those having a potency in one of theassays described herein (including the food intake, gastric emptying,pancreatic secretion, weight reduction or body composition assays) whichis greater than the potency of PP, NPY, PYY, or PYY(3-36) in that sameassay.

In addition to the amelioration of hypertension in subjects in needthereof as a result of reduced food intake, weight loss, or treatingobesity, compounds of the invention may be used to treat hypotension asdescribed in Example 4.

Compounds of the invention may also be useful for potentiating,inducing, enhancing or restoring glucose responsivity in pancreaticislets or cells. These actions may be useful for treating or preventingconditions associated with metabolic disorders such as those describedabove and in U.S. patent application no. US20040228846. Assays fordetermining such activity are known in the art. For example, inpublished U.S. patent application no. US20040228846 (incorporated byreference in its entirety), assays are described for islet isolation andculture as well as determining fetal islet maturation. In the examplesof patent application US20040228846, intestine-derived hormone peptidesincluding pancreatic polypeptide (PP), neuropeptide Y (NPY),neuropeptide K (NPK), PYY, secretin, glucagon-like peptide-1 (GLP-1) andbombesin were purchased from Sigma. Collagenase type XI was obtainedfrom Sigma. RPMI 1640 culture medium and fetal bovine serum wereobtained from Gibco. A radioimmunoassay kit containing anti-insulinantibody ([¹²⁵I]-RIA kit) was purchased from Linco, St Louis.

Post-partum rat islets were obtained from P-02 year old rats. Adult ratislets were obtained from 6-8 week old rats. Fetal rat islets wereobtained as follows. Pregnant female rats were sacrificed on pregnancyday e21. Fetuses were removed front the uterus. 10-14 pancreata weredissected from each litter and washed twice in Hanks buffer. Thepancreata were pooled, suspended in 6 ml 1 mg/ml collagenase (Type XI,Sigma) and incubated at 37° C. for 8-10 minutes with constant shaking.The digestion was stopped by adding 10 volumes of ice-cold Hanks bufferfollowed by three washes with Hanks buffer. The islets were thenpurified by Ficoll gradient and cultured in 10% fetal bovine serum(FBS)/RPMI medium with or without addition of 1 μM IBMX. At the end offive days, 20 islets were hand picked into each tube and assayed forstatic insulin release. Generally, islets were first washed with KRPbuffer and then incubated with 1 ml of KRP buffer containing 3 mM (low)glucose for 30 minutes at 37° C. with constant shaking. After collectingthe supernatant, the islets were then incubated with 17 mM (high)glucose for one hour at 37° C. The insulin released from low or highglucose stimulation were assayed by radioimmunoassay (RIA) using the[¹²⁵I]-RIA kit. E21 fetal islets were cultured for 5 days in thepresence of 200 ng/ml PYY, PP, CCK, NPK, NPY, Secretin, GLP-1 orBombesin.

An exemplary in vivo assay is also provided using the Zucker DiabeticFatty (ZDF) male rat, an inbred (>F30 Generations) rat model thatspontaneously expresses diabetes in all fa/fa males fed a standardrodent diet Purina 5008. In ZDF fa-fa males, hyperglycemia begins todevelop at about seven weeks of age and glucose levels (fed) typicallyreach 500 mg/DL by 10 to 11 weeks of age. Insulin levels (fed) are highduring the development of diabetes. However, by 19 weeks of age insulindrops to about the level of lean control litter mates. Plasmatriglyceride and cholesterol levels of obese rats are normally higherthan those of leans. In the assay, three groups of 7-week old ZDF rats,with 6 rats per group, received the infusion treatment by ALZA pump for14 days: 1) vehicle control, 2) and 3), PYY with two different doses,100 pmol/kg/hr and 500 pmol/kg/hr respectively. Four measurements weretaken before the infusion and after the infusion at day 7 and day 14: 1)plasma glucose level. 2) plasma insulin level, and 3) plasmatriglycerides (TG) level, as well as oral glucose tolerance (OGTT) test.Accordingly, these assays can be used with compounds of the invention totest for desired activity.

Other uses contemplated for the PPF polypeptides include methods forreducing aluminum (Al) concentrations in the central nervous system (seeU.S. Pat. No. 6,734,166, incorporated by reference in its entirety) fortreating, preventing, or delay the onset of Alzheimer's disease. Assaysfor determining effects on Al are known in the art and can be found inU.S. Pat. No. 6,734,166 using diploid and Ts mice. These mice wereindividually housed in Nalgene® brand metabolism or polypropylene cagesand given three days to adjust to the cages before experimentation. Micehad free access to food (LabDiet® NIH Rat and Moust/Auto 6FSK52, St.Louis, Mo.) and water during the experiment except for the 16 hoursprior to euthanasia when no food was provided. Mice were given dailysubcutaneous injections of either active compound or saline. Mice weresacrificed at the end of day 13 for one experiment and day 3 foranother, and samples were collected. Mice brain samples were weighted inclean teflon liners and prepared for analysis by microwave digestion inlow trace element grade nitric acid. Sample were then analyzed for Alcontent using Inductively Coupled Plasma Mass Spectrometry (Nuttall etal., Annals of Clinical and Laboratory Science 25, 3, 264-271 (1995)).All tissue handling during analysis took place in a clean roomenvironment utilizing HEPA air filtration systems to minimize backgroundcontamination.

The compounds of the invention exhibit a broad range of biologicalactivities, some related to their antisecretory and antimotilityproperties. The compounds may suppress gastrointestinal secretions bydirect interaction with epithelial cells or, perhaps, by inhibitingsecretion of hormones or neurotransmitters which stimulate intestinalsecretion. Anti-secretory properties include inhibition of gastricand/or pancreatic secretions and can be useful in the treatment orprevention of diseases and disorders including gastritis, pancreatitis,Barrett's esophagus, and Gastroesophageal Reflux Disease.

Compounds of the invention are useful in the treatment of any number ofgastrointestinal disorders (see e.g., Harrison's Principles of InternalMedicine, McGraw-Hill Inco, N.Y., 12th Ed.) that are associated withexcess intestinal electrolyte and water secretion as well as decreasedabsorption, e.g., infectious diarrhea, inflammatory diarrhea, shortbowel syndrome, or the diarrhea which typically occurs followingsurgical procedures, e.g., ileostomy. Examples of infectious diarrheainclude, without limitation, acute viral diarrhea, acute bacterialdiarrhea (e.g., salmonella, campylobacter, and clostridium or due toprotozoal infections), or traveler's diarrhea (e.g., Norwalk virus orrotavirus). Examples of inflammatory diarrhea include, withoutlimitation, malabsorption syndrome, tropical sprue, chronicpancreatitis, Crohn's disease, diarrhea, and irritable bowel syndrome.It has also been discovered that the peptides of the invention can beused to treat an emergency or life-threatening situation involving agastrointestinal disorder, e.g., after surgery or due to cholera.

Compounds of the invention may also be useful for treating or preventingintestinal damage as opposed to merely treating the symptoms associatedwith the intestinal damage (for example, diarrhea). Such damage to theintestine may be, or a result of, ulcerative colitis, inflammatory boweldisease, bowel atrophy, loss bowel mucosa, and/or loss of bowel mucosalfunction (see WO 03/105763, incorporated herein by reference in itsentirety). A simple and reproducible rat model of chronic colonicinflammation has been previously described by Morris G P. et al.,“Hapten-induced model of chronic inflammation and ulceration in the ratcolon.” Gastroenterology. 1989; 96:795-803. It exhibits a relativelylong duration of inflammation and ulceration, affording an opportunityto study the pathophysiology of colonic inflammatory disease in aspecifically controlled fashion, and to evaluate new treatmentspotentially applicable to inflammatory bowel disease in humans.

Assays for such activity, as described in WO 03/105763, include 11 weekold male HSD rats, ranging 250-300 grams housed in a 12:12 light:darkcycle, and allowed ad libitum access to a standard rodent diet (TekladLM 485, Madison, Wis.) and water. The animals were fasted for 24 hoursbefore the experiment. Rats were anesthetized with 3% isoflorane andplaced on a regulated heating pad set at 37° C. A gavage needle wasinserted rectally into the colon 7 cm. The haptentrinitrobenzenzenesulfonic acid (TNBS) dissolved in 50% ethanol (v/v)was delivered into the lumen of the colon through the gavage needle at adose of 30 mg/kg, in a total volume of 0 0.4-0.6 mL, as described inMazelin, et al., “Protective role of vagal afferents inexperimentally-induced colitis in rats.” Juton Nerv Syst. 1998; 73:3845. Control groups received saline solution (NaCl 0.9%)intracolonically.

Four days after induction of colitis, the colon was resected fromanesthetized rats, which were then euthanized by decapitation. Weightsof excised colon and spleen were measured, and the colons photographedfor scoring of gross morphologic damage. Inflammation was defined asregions of hyperemia and bowel wall thickening.

Compounds of the invention may also be used to treat or preventpancreatic tumors (e.g., inhibit the proliferation of pancreatictumors). Methods of the invention include reducing the proliferation oftumor cells. The types of benign pancreatic tumor cells which may betreated in accordance with the present invention include serous cystadenomas, microcystic tumors, and solid-cystic tumors. The method isalso effective in reducing the proliferation of malignant pancreatictumor cells such as carcinomas arising from the ducts, acini, or isletsof the pancreas. U.S. Pat. No. 5,574,010 (incorporated by reference inits entirety) provides exemplary assays for testing anti-proliferativeproperties. For example, the '010 patent provides that PANC-1 andMiaPaCa-2 are two human pancreatic adenocarcinoma cancer cell lineswhich are available commercially from suppliers such as American TypeCulture Collection, ATCC (Rockville, Md.). The two tumor cells weregrown in RPMI-1640 culture media supplemented with 10% fetal bovineserum, 29.2 mg/L of glutamine, 25 μg gentamicin, 5 ml penicillin,streptomycin, and fungizone solution (JRH Biosciences Lenexa, Kans.) at37 degrees Celcius in a NAPCO water jacketed 5% CO₂ incubator. All celllines were detached with 0.25% trypsin (Clonetics, San Diego, Calif.)once to twice a week when a confluent monolayer of tumor cells wasachieved. Cells were pelleted for 7 minutes at 500 g in a refrigeratedcentrifuge at 4 degrees Celcius, and resuspended in trypsin freefortified RPMI 1640 culture media. Viable cells were counted on ahemocytometer slide with trypan blue.

Ten thousand, 20,000, 40,000 and 80,000 cells of each type were added to96 well microculture plates (Costar, Cambridge. Mass.) in a total volumeof 200 ul of culture media per well. Cells were allowed to adhere for 24hours prior to addition of the PYY or test peptide. Fresh culture mediawas exchanged prior to addition of peptides. In vitro incubation ofpancreatic tumor cells with either PYY or test compound was continuedfor 6 hours and 36 hours in length. PYY was added to cells at doses of250 pmol, 25 pmol, and 2.5 pmol per well (N=14). Test compound was addedto cells cultures at doses of 400 pmol, 40 pmol, and 4 pmol per well.Control wells received 2 ul of 0.9% saline to mimic the volume andphysical disturbance upon adhered tumor cells. Each 96 well platecontained 18 control wells to allow for comparison within each plateduring experimentation. Ninety-six (96) well plates were repeated 6times with varying concentrations of PYY and test compound in both thePANC-1 and MiaPaCa-2 cells.

At the end of the incubation period,3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium Bromide, MTTtetrazolium bromide (Sigma, St. Louis, Mo.) was added to fresh culturemedia at 0.5 mg/ml. Culture media was exchanged and tumor cells wereincubated for 4 hours with MTT tetrazolium bromide at 37 degreesCelcius. At the end of incubation, culture media was aspirated. Formazoncrystal precipitates were dissolved in 200 ul of dimethyl sulfoxide(Sigma, St. Louis, Mo.). Quantitation of solubilized formazon wasperformed by obtaining absorption readings at 500 nm wavelength on anELISA reader (Molecular Devices, Menlo Park, Calif.). The MTT assaymeasures mitochondrial NADH dependent dehydrogenase activity, and it hasbeen among the most sensitive and reliable method to quantitative invitro chemotherapy responses of tumor cells. (Alley, M. C., Scadiero, D.A., Monk, A., Hursey, M. L., Dzerwinski, M. J., Fine, D. L., Abbott, B.J., Mayo, J. G., Shoemaker, R. H. and Boyd, M. R., Feasibility of drugscreening with panels of human tumor cell lines using a microculturetetrazolium assay Cancer Res., 48:589-601, 1988; Carmichael, J.,DeGraff, W. G., Gazdar. A. F. Minna, J. D. and Mitchell, J. B.,Evaluation of a tetrazolium-based semiautomated colorimetric assay:Assessment of chemosensitivity testing. Cancer Res., 47:936-942, 1987;McHale, A. P., McHale. L., Use of a tetrazolium based colorimetric assayin assessing photoradiation therapy in vitro. Cancer Lett. 41:315-321,1988; and Saxton, R. E., Huang, M. Z., Plante D., Fetterman, H. F.,Lutkin, R. B., Soudant. J., Castro, D. J., Laser and daunomycinchemophototherapy of human carcinoma cells. J. Clin. Laser Med. andSurg., 10(5):331-336, 1992.) Analysis of absorption readings at 550 nmwere analyzed by grouping wells of the same test conditions andverifying differences occurring between control and the various peptideconcentration treatments by one-way ANOVA.

An exemplary in vivo assay is also provided. The human pancreatic ductaladenocarcinoma Mia Paca-2 was examined for in vivo growth inhibition bypeptide YY and test compound. Seventy thousand to 100,000 human MiaPaCa-2 cells were orthotopically transplanted into 48 male athymic mice.After one week, the animals were treated with either PYY or testcompound at 200 pmol/kg/hr via mini-osmotic pumps for four weeks. Thepaired cultures received saline. At sacrifice, both tumor size and masswere measured. Control mice had significant human cancer growth withinthe pancreas as evidenced by histologic sections. At 9 weeks, ninetypercent (90%) of control mice had substantial metastatic disease. Tumormass was decreased by 60.5% in test treated mice and 27% in PYY treatedmice.

PPF polypeptides may be administered alone or in combination withpharmaceutically acceptable carriers or excipients, in either single ormultiple doses. These pharmaceutical compounds may be formulated withpharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients in accordance with conventionaltechniques such as those disclosed in Remington's PharmaceuticalSciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A.“Parenteral Formulations of Proteins and Peptides: Stability andStabilizers.” Journal of Parenteral Science and Technology, TechnicalReport No. 10, Supp. 42:2S (1988), incorporated by reference.

The PPF polypeptides may be provided in dosage unit form. For example,therapeutically effective amounts of the PPF polypeptide for affectingbody composition will vary with many factors including the age andweight of the patient, the patient's physical condition, their use incombination with other treatments, the ultimate goal that is to beachieved, such as overall weight loss and/or maintaining or increasinglean body mass, as well as other factors. However, typical doses maycontain from a lower limit of about 0.05 μg, about 0.1 μg, about 1 μg,about 5 μg, about 10 μg, about 50 μg, about 75 μg or about 100 μg, to anupper limit of about 50 μg, about 100 μg, about 500 μg, about 1 mg,about 5 mg, about 10 mg, about 15 mg, about 50 mg, about 100 mg or about150 mg of the pharmaceutical compound per day. Also contemplated areother dose ranges such as 0.1 μg to 1 mg of the compound per dose, or atabout 0.001 μg/kg to about 500 μg/kg per dose. In some embodiments, thePPF polypeptide of the invention is administered peripherally at a doseof about 0.5 μg to about 5 mg per day in single or divided doses orcontrolled continual release, or at about 0.01 μg/kg to about 500 μg/kgper dose, or at about 0.05 μg/kg to about 250 μg/kg. In someembodiments, the PPF polypeptide is administered at a dose below about50 μg/kg. Dosages in these ranges will vary with the potency of eachanalog or derivative, of course, and may be readily determined by one ofskill in the art.

The doses per day may be delivered in discrete unit doses, providedcontinuously in a 24 hour period or any portion of that the 24 hours.The number of doses per day may be from 1 to about 4 per day, althoughit could be more. Continuous delivery can be in the form of a continuousinfusion. Other contemplated exemplary doses and infusion rates includefrom 0.005 nmol/kg to about 20 nmol/kg per discrete dose or from about0.01/pmol/kg/min to about 10 pmol/kg/min in a continuous infusion. Thesedoses and infusions can be delivered by any known conventional orfuture-developed peripheral method, e.g., intravenous (i.v.),intradermal, intramuscular, intramammary, intraperitoneal, intrathecal,retrobulbar, intrapulmonary (e.g., term release); subcutaneousadministration (s.c.), by oral, sublingual, nasal, anal, vaginal, ortransdermal delivery, or by surgical implantation at a particular site.Exemplary total dose/delivery of the pharmaceutical composition giveni.v. may be about 1 μg to about 8 μg per day, whereas totaldose/delivery of the pharmaceutical composition given s.c. may be about6 μg to about 16 mg per day.

In one general aspect, methods of the invention may include the use ofother body weight or body fat regulating compounds in combination with aPPF polypeptide. In the methods of the present invention, a PYY, PYYagonist or PPF polypeptide of the invention may be administeredseparately or together with one or more other compounds and compositionsthat exhibit a long term or short-term action to reduce nutrientavailability, food intake, body weight, body weight gain or to alterbody composition, for example. Such compounds include, but are notlimited to, other compounds and compositions that comprise an amylin,amylin agonist or amylin analog agonist, salmon calcitonin, acholecystokinin (CCK) or CCK agonist, a leptin (OB protein) or leptinagonist, an exendin or exendin analog agonist, a glucagon-like peptide-1(GLP-1), GLP-1 agonist or GLP-1 analog agonist, CCK, CCK agonists,calcitonin, calcitonin agonists, small molecule cannabinoid CB1 receptorantagonists, rimonabant, 11 beta-hydroxysteroid dehydrogenase-1inhibitors, sibutramine, phentermine and other drugs marketed for thetreatment of obesity, such as appetite control. These compounds may beadministered in combination, simultaneously or sequentially. Suitableamylin agonists include, for example, [^(25,28,29)Pro-] human amylin(also known as “pramlintide,” and described in U.S. Pat. Nos. 5,686,511and 5,998,367) and salmon calcitonin. In some embodiments, the CCK usedis CCK octapeptide (CCK-8). Leptin is discussed in, for example,(Pelleymounter et al., Science 269: 540-3 (1995); Halaas et al., Science269: 543-6 (1995); Campfield et al., Science 269: 546-9 (1995)).Suitable exendins include exendin-3 and exendin-4, and excendin agonistcompounds include, for example, those described in PCT Publications WO99/07404, WO 99/25727, and WO 99/25728.

Polypeptide Production and Purification

The PPF polypeptides described herein may be prepared using standardrecombinant techniques or chemical peptide synthesis techniques known inthe art, e.g., using an automated or semi-automated peptide synthesizer,or both.

The PPF polypeptides of the invention can be synthesized in solution oron a solid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. See, e.g. Stewart and Young, SolidPhase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tam etal., J. Am. Chem. Soc. 105: 6442 (1983); Merrifield, Science 232: 341-7(1986); and Barany and Merrifield, The Peptides, Gross and Meienhoter,eds., Academic Press, New York, 1-284 (1979). Solid phase peptidesynthesis may be carried out with an automatic peptide synthesizer(e.g., Model 430A, Applied Biosystems Inc., Foster City, Calif.) usingthe NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (see, AppliedBiosystems User's Manual for the ABI 430A Peptide Synthesizer, Version1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc.,Foster City, Calif.) with capping. Peptides may also be assembled usingan Advanced ChemTech Synthesizer (Model MPS 350, Louisville, Ky.).Peptides may be purified by RP-HPLC (preparative and analytical) using,e.g., a Waters Delta Prep 3000 system and a C4, C8, or C18 preparativecolumn (10μ, 2.2×25 cm; Vydac, Hesperia, Calif.). The active protein canbe readily synthesized and then screened in screening assays designed toidentify reactive peptides.

The PPF polypeptides of the present invention may alternatively beproduced by recombinant techniques well known in the art. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor (1989). These PYY analog polypeptides produced byrecombinant technologies may be expressed from a polynucleotide. Oneskilled in the art will appreciate that the polynucleotides, includingDNA and RNA, that encode such encoded PYY analog polypeptides may beobtained from the wild-type PYY cDNA, taking into consideration thedegeneracy of codon usage. These polynucleotide sequences mayincorporate codons facilitating transcription and translation of mRNA inmicrobial hosts. Such manufacturing sequences may readily be constructedaccording to the methods well known in the art. See, e.g., WO 83/04053.The polynucleotides above may also optionally encode an N-terminalmethionyl residue. Non-peptide compounds useful in the present inventionmay be prepared by art-known methods. For example, phosphate-containingamino acids and peptides containing such amino acids may be preparedusing methods known in the art. See, e.g., Bartlett and Landen, Bioorg.Chem. 14: 356-77 (1986).

A variety of expression vector/host systems may be utilized to containand express a PPF polypeptide coding sequence. These include but are notlimited to microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransfected with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.Mammalian cells that are useful in recombinant protein productionsinclude but are not limited to VERO cells, HeLa cells, Chinese hamsterovary (CHO) cell lines, COS cells (such as COS-7), WI 38, BHK, HepG2,3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols forthe recombinant expression of the protein are described herein below.

As such, polynucleotide sequences provided by the invention are usefulin generating new and useful viral and plasmid DNA vectors, new anduseful transformed and transfected procaryotic and eucaryotic host cells(including bacterial, yeast, and mammalian cells grown in culture), andnew and useful methods for cultured growth of such host cells capable ofexpression of the present PPF polypeptides. The polynucleotide sequencesencoding PPF polypeptides herein may be useful for gene therapy ininstances where underproduction of PP, PYY, or NPY would be alleviated,or the need for increased levels of such would be met.

The present invention also provides for processes for recombinant DNAproduction of the present PPF polypeptides. Provided is a process forproducing the PPF polypeptides from a host cell containing nucleic acidsencoding such PPF polypeptides comprising: (a) culturing said host cellcontaining polynucleotides encoding such PPF polypeptides underconditions facilitating the expression of such DNA molecule; and (b)obtaining such PPF polypeptides.

Host cells may be prokaryotic or eukaryotic and include bacteria,mammalian cells (such as Chinese Hamster Ovary (CHO) cells, monkeycells, baby hamster kidney cells, cancer cells or other cells), yeastcells, and insect cells.

Mammalian host systems for the expression of the recombinant proteinalso are well known to those of skill in the art. Host cell strains maybe chosen for a particular ability to process the expressed protein orproduce certain post-translation modifications that will be useful inproviding protein activity. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing, which cleaves a “prepro” form of theprotein, may also be important for correct insertion, folding and/orfunction. Different host cells, such as CHO, HeLa, MDCK, 293, W138, andthe like, have specific cellular machinery and characteristic mechanismsfor such post-translational activities, and may be chosen to ensure thecorrect modification and processing of the introduced foreign protein.

Alternatively, a yeast system may be employed to generate the PPFpolypeptides of the present invention. The coding region of the PPFpolypeptide cDNA is amplified by PCR. A DNA encoding the yeastpre-pro-alpha leader sequence is amplified from yeast genomic DNA in aPCR reaction using one primer containing nucleotides 1-20 of the alphamating factor gene and another primer complementary to nucleotides255-235 of this gene (Kurjan and Herskowitz, Cell, 30: 933-43 (1982)).The pre-pro-alpha leader coding sequence and PPF polypeptide codingsequence fragments are ligated into a plasmid containing the yeastalcohol dehydrogenase (ADH2) promoter, such that the promoter directsexpression of a fusion protein consisting of the pre-pro-alpha factorfused to the mature PPF polypeptide. As taught by Rose and Broach, Meth.Enz. 185: 234-79, Goeddel ed., Academic Press, Inc., San Diego, Calif.(1990), the vector further includes an ADH2 transcription terminatordownstream of the cloning site, the yeast “2-micron” replication origin,the yeast leu-2d gene, the yeast REP1 and REP2 genes, the E. coliβ-lactamase gene, and an E. coli origin of replication. The β-lactamaseand leu-2d genes provide for selection in bacteria and yeast,respectively. The leu-2d gene also facilitates increased copy number ofthe plasmid in yeast to induce higher levels of expression. The REP1 andREP2 genes encode proteins involved in regulation of the plasmid copynumber.

The DNA construct described in the preceding paragraph is transformedinto yeast cells using a known method, e.g., lithium acetate treatment(Steams et al., Meth. Enz. 185: 280-97 (1990)). The ADH2 promoter isinduced upon exhaustion of glucose in the growth media (Price et al.,Gene 55: 287 (1987)). The pre-pro-alpha sequence effects secretion ofthe fusion protein from the cells. Concomitantly, the yeast KEX2 proteincleaves the pre-pro sequence from the mature PYY analog polypeptides(Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330-4 (1984)).

PPF polypeptides of the invention may also be recombinantly expressed inyeast using a commercially available expression system, e.g., the PichiaExpression System (Invitrogen, San Diego, Calif.), following themanufacturer's instructions. This system also relies on thepre-pro-alpha sequence to direct secretion, but transcription of theinsert is driven by the alcohol oxidase (AOXI) promoter upon inductionby methanol. The secreted PPF polypeptide is purified from the yeastgrowth medium by, e.g., the methods used to purify PPF polypeptide frombacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding PYY analog polypeptides may be clonedinto the baculovirus expression vector pVL1393 (PharMingen, San Diego,Calif.). This PPF polypeptides-containing vector is then used accordingto the manufacturer's directions (PharMingen) to infect Spodopterafrugiperda cells in sF9 protein-free media and to produce recombinantprotein. The protein is purified and concentrated from the media using aheparin-Sepharose column (Pharmacia, Piscataway, N.J.) and sequentialmolecular sizing columns (Amicon, Beverly, Mass.), and resuspended inPBS. SDS-PAGE analysis shows a single band and confirms the size of theprotein, and Edman sequencing on a Proton 2090 Peptide Sequencerconfirms its N-terminal sequence.

For example, the DNA sequence encoding the predicted mature PYY analogpolypeptide may be cloned into a plasmid containing a desired promoterand, optionally, a leader sequence (see, e.g., Better et al., Science240: 1041-3 (1988)). The sequence of this construct may be confirmed byautomated sequencing. The plasmid is then transformed into E. coli,strain MC1061, using standard procedures employing CaCl2 incubation andheat shock treatment of the bacteria (Sambrook et al., supra). Thetransformed bacteria are grown in LB medium supplemented withcarbenicillin, and production of the expressed protein is induced bygrowth in a suitable medium. If present, the leader sequence will affectsecretion of the mature PYY analog polypeptide and be cleaved duringsecretion. The secreted recombinant protein is purified from thebacterial culture media by the method described herein below.

Alternatively, the PPF polypeptides of the invention may be expressed inan insect system. Insect systems for protein expression are well knownto those of skill in the art. In one such system, Autographa californicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.The PPF polypeptide coding sequence is cloned into a nonessential regionof the virus, such as the polyhedrin gene, and placed under control ofthe polyhedrin promoter. Successful insertion of PYY analog polypeptidewill render the polyhedrin gene inactive and produce recombinant viruslacking coat protein coat. The recombinant viruses are then used toinfect S. frugiperda cells or Trichoplusia larvae in which PYY analogpolypeptide is expressed (Smith et al., J. Virol. 46: 584 (1983);Engelhard et al., Proc. Natl. Acad. Sci. USA 91: 3224-7 (1994)).

In another example, the DNA sequence encoding the PPF polypeptide may beamplified by PCR and cloned into an appropriate vector, for example,pGEX-3X (Pharmacia, Piscataway, N.J.). The pGEX vector is designed toproduce a fusion protein comprising glutathione-S-transferase (GST),encoded by the vector, and a protein encoded by a DNA fragment insertedinto the vector's cloning site. The primers for the PCR may be generatedto include, for example, an appropriate cleavage site. The recombinantfusion protein may then be cleaved from the GST portion of the fusionprotein. The pGEX-3X/PYY analog polypeptide construct is transformedinto E. coli XL-Blue cells (Stratagene, La Jolla, Calif.), andindividual transformants are isolated and grown at 37° C. in LB medium(supplemented with carbenicillin) to an optical density at wavelength600 nm of 0.4, followed by further incubation for 4 hours in thepresence of 0.5 mM Isopropyl β-D-Thiogalactopyranoside (Sigma ChemicalCo., St. Louis, Mo.). Plasmid DNA from individual transformants ispurified and partially sequenced using an automated sequencer to confirmthe presence of the desired PPF polypeptide-encoding gene insert in theproper orientation.

The fusion protein, expected to be produced as an insoluble inclusionbody in the bacteria, may be purified as follows. Cells are harvested bycentrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; andtreated with 0.1 mg/mL lysozyme (Sigma Chemical Co.) for 15 min. at roomtemperature. The lysate is cleared by sonication, and cell debris ispelleted by centrifugation for 10 min. at 12,000×g. The fusionprotein-containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mMEDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000×g.The pellet is resuspended in standard phosphate buffered saline solution(PBS) free of Mg⁺⁺ and Ca⁺⁺. The fusion protein is further purified byfractionating the resuspended pellet in a denaturing SDS polyacrylamidegel (Sambrook et al., supra). The gel is soaked in 0.4 M KCl tovisualize the protein, which is excised and electrocuted in gel-runningbuffer lacking SDS. If the GST/PYY analog polypeptide fusion protein isproduced in bacteria as a soluble protein, it may be purified using theGST Purification Module (Pharmacia Biotech).

The fusion protein may be subjected to digestion to cleave the GST fromthe mature PYY analog polypeptide. The digestion reaction (20-40 μgfusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 mLPBS) is incubated 16-48 hrs. at room temperature and loaded on adenaturing SDS-PAGE gel to fractionate the reaction products. The gel issoaked in 0.4 M KCl to visualize the protein bands. The identity of theprotein band corresponding to the expected molecular weight of the PYYanalog polypeptide may be confirmed by partial amino acid sequenceanalysis using an automated sequencer (Applied Biosystems Model 473A,Foster City, Calif.).

In one method of recombinant expression of the PPF polypeptides of thepresent invention, HEK 293 cells may be co-transfected with plasmidscontaining the PYY analog polypeptide cDNA in the pCMV vector (5′ CMVpromoter, 3′ HGH poly A sequence) and pSV2neo (containing the neoresistance gene) by the calcium phosphate method. In some embodiments,the vectors should be linearized with Seal prior to transfection.Similarly, an alternative construct using a similar pCMV vector with theneo gene incorporated can be used. Stable cell lines are selected fromsingle cell clones by limiting dilution in growth media containing 0.5mg/mL G418 (neomycin-like antibiotic) for 10-14 days. Cell lines arescreened for PYY analog polypeptide expression by ELISA or Western blot,and high-expressing cell lines are expanded for large scale growth.

In some embodiments, the transformed cells are used for long-term,high-yield protein production and stable expression may be desirable.Once such cells are transformed with vectors that contain selectablemarkers along with the desired expression cassette, the cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The selectable marker is designed to conferresistance to selection, and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Resistantclumps of stably transformed cells can be proliferated using tissueculture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that havebeen transformed for recombinant protein production. Such selectionsystems include, but are not limited to, HSV thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside, G418; also, that confers resistance tochlorsulfuron; and hygro, that confers resistance to hygromycin.Additional selectable genes that may be useful include trpB, whichallows cells to utilize indole in place of tryptophan, or hisD, whichallows cells to utilize histinol in place of histidine. Markers thatgive a visual indication for identification of transformants includeanthocyanins, β-glucuronidase and its substrate, GUS, and luciferase andits substrate, luciferin.

Many of the PPF polypeptides of the present invention may be producedusing a combination of both automated peptide synthesis and recombinanttechniques. For example, a PPF polypeptide of the present invention maycontain a combination of modifications including deletion, substitution,and insertion by PEGylation. Such a PPF polypeptide may be produced instages. In the first stage, an intermediate PPF polypeptide containingthe modifications of deletion, substitution, insertion, and anycombination thereof, may be produced by recombinant techniques asdescribed. Then after an optional purification step as described below,the intermediate PPF polypeptide is PEGylated through chemicalmodification with an appropriate PEGylating reagent (e.g., from NeKtarTherapeutics, San Carlos, Calif.) to yield the desired PPF polypeptide.One skilled in the art will appreciate that the above-describedprocedure may be generalized to apply to a PPF polypeptide containing acombination of modifications selected from deletion, substitution,insertion, derivation, and other means of modification well known in theart and contemplated by the present invention.

It may be desirable to purify the PPF polypeptides generated by thepresent invention. Peptide purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography, polyacrylamidegel electrophoresis, and isoelectric focusing. A particularly efficientmethod of purifying peptides is reverse phase HPLC, followed bycharacterization of purified product by liquid chromatography/massspectrometry (LC/MS) and Matrix-Assisted Laser Desorption Ionization(MALDI) mass spectrometry. Additional confirmation of purity is obtainedby determining amino acid analysis.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified peptide” as used herein, isintended to refer to a composition, isolatable from other components,wherein the peptide is purified to any degree relative to its naturallyobtainable state. A purified peptide therefore also refers to a peptide,free from the environment in which it may naturally occur.

Generally, “purified” will refer to a peptide composition that has beensubjected to fractionation to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the peptide forms the major component ofthe composition, such as constituting about 50%, about 60%, about 70%,about 80%, about 90%, about 95% or more of the peptides in thecomposition.

Various techniques suitable for use in peptide purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies, and the like;heat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the peptides always be provided intheir most purified state. Indeed, it is contemplated that lesssubstantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed, utilizing anHPLC apparatus, will generally result in a greater “-fold” purificationthan the same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

One may optionally purify and isolate such PPF polypeptides from othercomponents obtained in the process. Methods for purifying a polypeptidecan be found in U.S. Pat. No. 5,849,883. These documents describespecific exemplary methods for the isolation and purification of G-CSFcompositions that may be useful in isolating and purifying the PPFpolypeptides of the present invention. Given the disclosure of thesepatents, it is evident that one of skill in the art would be well awareof numerous purification techniques that may be used to purify PPFpolypeptides from a given source.

Also it is contemplated that a combination of anion exchange andimmunoaffinity chromatography may be employed to produce purified PPFpolypeptide compositions of the present invention.

Pharmaceutical Compositions

The present invention also relates to pharmaceutical compositionscomprising a therapeutically or prophylactically effective amount of atleast one PPF polypeptide of the invention, or a pharmaceuticallyacceptable salt thereof, together with pharmaceutically acceptablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers useful in the delivery of the PPF polypeptides. Suchcompositions may include diluents of various buffer content (e.g.,acetate, citrate, glutamate, tartrate, phosphate, TRIS), pH and ionicstrength; additives such as surfactants and solubilizing agents (e.g.,sorbitan monooleate, lecithin, Pluronics, Tween 20 & 80, Polysorbate 20& 80, propylene glycol, ethanol, PEG-40, sodium dodecyl sulfate),anti-oxidants (e.g., monothioglyercol, ascorbic acid, acetylcysteine,sulfurous acid salts (bisulfise and metabisulfite), preservatives (e.g.,phenol, meta-cresol, benzyl alcohol, parabens (methyl, propyl, butyl),benzalkonium chloride, chlorobutanol, thimersol, phenylmercuric salts,(acetate, borate, nitrate), and tonicity/bulking agents (glycerine,sodium chloride, mannitol, sucrose, trehalose, dextrose); incorporationof the material into particulate preparations of polymeric compounds,such as polylactic acid, polyglycolic acid, etc., or in association withliposomes. Such compositions will influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thepresent PPF polypeptides. See, e.g., Remington's Pharmaceutical Sciences1435-712, 18th ed., Mack Publishing Co., Easton, Pa. (1990).

In general, the present PPF polypeptides will be useful in the same waythat PP, PYY, or NPY is useful in view of their pharmacologicalproperties. One exemplary use is to peripherally administer such PPFpolypeptides for the treatment or prevention of metabolic conditions anddisorders. In particular, the compounds of the invention possessactivity as agents to reduce nutrient availability, reduce of foodintake, and effect weight loss.

The present PPF polypeptides may be formulated for peripheraladministration, including formulation for injection, oraladministration, nasal administration, pulmonary administration, topicaladministration, or other types of administration as one skilled in theart will recognize. More particularly, administration of thepharmaceutical compositions according to the present invention may bevia any common route so long as the target tissue is available via thatroute. In some embodiments, the pharmaceutical compositions may beintroduced into the subject by any conventional peripheral method, e.g.,by intravenous, intradermal, intramuscular, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary (e.g., termrelease); by oral, sublingual, nasal, anal, vaginal, or transdermaldelivery, or by surgical implantation at a particular site. Thetreatment may consist of a single dose or a plurality of doses over aperiod of time. Controlled continual release of the compositions of thepresent invention is also contemplated.

The formulation may be composed in various forms, e.g., solid, liquid,semisolid or liquid. The formulation may be liquid or may be solid, suchas lyophilized, for reconstitution. The term “solid,” as used herein, ismeant to encompass all normal uses of this term including, for example,powders and lyophilized formulations. Aqueous compositions of thepresent invention comprise an effective amount of the PPF polypeptide,dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium. The phrase “pharmaceutically or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions. In some cases, it will be convenient to provide a PPFpolypeptide and another food-intake-reducing, plasma glucose-lowering orplasma lipid-altering agent, such as an amylin, an amylin agonistanalog, a CCK or CCK agonist, or a leptin or leptin agonist, or anexendin or exendin agonist analog, and small molecule cannabinoid CB1receptor antagonists, rimonabant, beta-hydroxysteroid dehydrogenase-1inhibitors, sibutramine, phentermine and other drugs marketed fortreatment of obesity in a single composition or solution foradministration together. In other cases, it may be more advantageous toadminister the additional agent separately from said PPF polypeptide.

The PPF polypeptide of the invention may be prepared for administrationas solutions of free base, or pharmacologically acceptable salts inwater suitably mixed with surface active agents (e.g., sorbitanmonooleate, polyoxyethylene sorbitan monolaurate (Tween 20),polyoxyethylene sorbitan monooleate (Tween 80), lecithin,polyoxyethylene-polyoxypropylene copolymers (Pluronics),hydroxypropylcellulose) or complexation agents (e.g.,hydroxypropyl-b-cyclodextrin, sulfobutyether-h-cyclodextrin (Captisol),polyvinylpyrrolidone). Pharmaceutically-acceptable salts include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups also can be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Such products are readily prepared by procedureswell known to those skilled in the art. Dispersions also can be preparedin glycerol, liquid polyethylene glycols, and mixtures thereof and inoils.

A preservative is, in the common pharmaceutical sense, a substance thatprevents or inhibits microbial growth and may be added to a formulationfor this purpose to avoid consequent spoilage of the formulation bymicroorganisms. While the amount of the preservative is not great, itmay nevertheless affect the overall stability of the peptide. Generally,under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms. While thepreservative for use in the pharmaceutical compositions can range from0.005 to 1.0% (w/v), in some embodiments, the range for eachpreservative, alone or in combination with others, is: benzyl alcohol(0.1-1.0%), or m-cresol (0.1-0.6%), or phenol (0.1-0.8%) or combinationof methyl (0.05-0.25%) and ethyl or propyl or butyl (0.005%-0.03%)parabens. The parabens are lower alkyl esters of para-hydroxybenzoicacid.

Surfactants can cause denaturation of protein, both of hydrophobicdisruption and by salt bridge separation. Relatively low concentrationsof surfactant may exert a potent denaturing activity, because of thestrong interactions between surfactant moieties and the reactive siteson proteins. However, judicious use of this interaction can stabilizeproteins against interfacial or surface denaturation. Surfactants whichcould further stabilize the peptide may optionally be present in therange of about 0.001 to 0.3% (w/v) of the total formulation and includepolysorbate 80 (i.e., polyoxyethylene(20) sorbitan monooleate), CHAPS®(i.e., 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate), Brij®(e.g., Brij 35, which is (polyoxyethylene (23) lauryl ether), poloxamer,or another non-ionic surfactant.

The stability of a peptide formulation of the present invention isenhanced by maintaining the pH of the formulation in the range of about3.0 to about 7.0 when in liquid form. In some embodiments, the PPFpolypeptide is suspended in an aqueous carrier, for example, in anbuffer solution at a pH of about 3.0 to about 8.0, about 3.5 to about7.4, about 3.5 to about 6.0, about 3.5 to about 5.0, about 3.7 to about4.7, about 3.7 to about 4.3 or about 3.8 to about 4.2. In someembodiments, parenteral formulations are isotonic or substantiallyisotonic. In some embodiments, the vehicle for parenteral products iswater. Water of suitable quality for parenteral administration can beprepared either by distillation or by reverse osmosis. Water may be usedas the aqueous vehicle for injection for use in the pharmaceuticalformulations. Useful buffers include sodium acetate/acetic acid, sodiumlactate/lactic acid, ascorbic acid, sodium citrate-citric acid, sodiumbicarbonate/carbonic acid, sodium succinate/succinic acid, Histidine,Sodium benzoate/benzoic acid, and sodium phosphates, andTris(hydroxymethyl)aminomethane. A form of repository or “depot” slowrelease preparation may be used so that therapeutically effectiveamounts of the preparation are delivered into the bloodstream over manyhours or days following transdermal injection or delivery.

In some embodiments, the pharmaceutical compositions of the presentinvention are formulated so as to be suitable for parenteraladministration, e.g., via injection or infusion. In some embodiments,liquid formulations are intended for parenteral administration. Suitableroutes of administration include intramuscular, intravenous,subcutaneous, intradermal, mucosal, intraarticular, intrathecal,bronchial and the like. These routes include, but are not limited to,oral, nasal, sublingual, pulmonary and buccal routes that may includeadministration of the PPF polypeptide in liquid, semi-solid or solidform. Administration via some routes require substantially more PPFpolypeptide to obtain the desired biological effects due to decreasedbioavailability compared to parenteral delivery. In addition, parenteralcontrolled release delivery can be achieved by forming polymericmicrocapsules, matrices, solutions, implants and devices andadministering them parenterally or by surgical means. Examples ofcontrolled release formulations are described in U.S. Pat. Nos.6,368,630, 6,379,704, and 5,766,627, which are incorporated herein byreference. These dosage forms may have a lower bioavailability due toentrapment of some of the peptide in the polymer matrix or device. Seee.g., U.S. Pat. Nos. 6,379,704, 6,379,703, and 6,296,842. In someembodiments, pharmaceutical compositions suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. In some embodiments, the form should be sterile and shouldbe fluid to the extent that is easily syringable. It is also desirablefor the PPF polypeptide of the invention to be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., sorbitol, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), dimethylacetamide, cremorphorEL, suitable mixtures thereof, and oils (e.g., soybean, sesame, castor,cottonseed, ethyl oleate, isopropyl myristate, glycofurol, corn). Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. The prevention ofthe action of microorganisms can be brought about by variousantibacterial an antifungal agents, for example, meta-cresol, benzylalcohol, parabens (methyl, propyl, butyl), chlorobutanol, phenol,phenylmercuric salts (acetate, borate, nitrate), sorbic acid,thimerosal, and the like. In some embodiments, tonicity agents (forexample, sugars, sodium chloride) may be included. Prolonged absorptionof the injectable compositions can be brought about by the use in thecompositions of agents delaying absorption (for example, aluminummonostearate and gelatin).

In some embodiments, for example non-parenteral formulations,sterilization may not be required. However, if sterilization is desiredor necessary, any suitable sterilization process can be used indeveloping the peptide pharmaceutical formulation of the presentinvention. Typical sterilization processes include filtration, steam(moist heat), dry heat, gases (e.g., ethylene oxide, formaldehyde,chlorine dioxide, propylene oxide, beta-propiolactone, ozone,chloropicrine, peracetic acid methyl bromide and the like), exposure toa radiation source, and aseptic handling. Filtration is the preferredmethod of sterilization for liquid formulations of the presentinvention. The sterile filtration involves filtration through 0.45 μmand 0.22 μm (1 or 2) which may be connected in series. After filtration,the solution is filled into appropriate vials or containers. Sterileinjectable solutions may be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle thatcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, exemplary methods ofpreparation are vacuum-drying and freeze-drying techniques that yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

In general, the PPF compounds may be formulated into a stable, safepharmaceutical composition for administration to a patient.Pharmaceutical formulations contemplated for use in the methods of theinvention may comprise from about 0.01 to about 20% (w/v), or from about0.05 to about 10%, of the PPF compound. The PPF compounds may be in anacetate, phosphate, citrate or glutamate buffer (for example, at a finalformulation concentration of from about 1-5 to about 60 mM) allowing apH of the final composition of about 3.0 to about 7.0 containingcarbohydrate or polyhydric alcohol as tonicity modifier and, optionally,approximately 0.005 to 5.0% (w/v) of a preservative selected from thegroup consisting of m-cresol, benzyl alcohol, methyl, ethyl, propyl andbutyl parabens and phenol. Such a preservative is generally included ifthe formulated peptide is to be included in a multiple use product.

Optionally, a stabilizer may be included in the present formulation. Ifincluded, however, a stabilizer useful in the practice of the presentinvention is a carbohydrate or a polyhydric alcohol. A suitablestabilizer useful in the practice of the present invention isapproximately 1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol.The polyhydric alcohols and carbohydrates share the same feature intheir backbones, i.e., —CHOH—CHOH—, which is responsible for stabilizingthe proteins. The polyhydric alcohols include such compounds assorbitol, mannitol, glycerol, and polyethylene glycols (PEGs). Thesecompounds are straight-chain molecules. The carbohydrates, such asmannose, ribose, sucrose, fructose, trehalose, maltose, inositol, andlactose, on the other hand, are cyclic molecules that may contain a ketoor aldehyde group. These two classes of compounds have been demonstratedto be effective in stabilizing protein against denaturation caused byelevated temperature and by freeze-thaw or freeze-drying processes.Suitable carbohydrates include: galactose, arabinose, lactose or anyother carbohydrate which does not have an adverse affect on a diabeticpatient (if this is a desirable property), i.e., the carbohydrate is notmetabolized to form unacceptably large concentrations of glucose in theblood.

In some embodiments, if a stabilizer is included, the PPF polypeptide isstabilized with a polyhydric alcohol such as sorbitol, mannitol,inositol, glycerol, xylitol, and polypropylene/ethylene glycolcopolymer, as well as various polyethylene glycols (PEG) of molecularweight 200, 400, 1450, 3350, 4000, 6000, and 8000). Another usefulfeature of the lyophilized formulations of the present invention is themaintenance of the tonicity of the lyophilized formulations describedherein with the same formulation component that serves to maintain theirstability. In some embodiments, mannitol is the polyhydric alcohol usedfor this purpose.

Containers may also be considered to be a part of the formulation of aninjection, for there is no container that is totally inert, or does notin some way affect the liquid it contains, particularly if the liquid isaqueous. Therefore, the selection of a container for a particularinjection must be based on a consideration of the composition of thecontainer, as well as of the solution, and the treatment to which itwill be subjected. If necessary, adsorption of the peptide to the glasssurface of the vial can also be minimized, by use of borosilicate glass,for example, Wheaton Type I borosilicate glass #33 (Wheaton Type I-33)or its equivalent (Wheaton Glass Co.). Other vendors of similarborosilicate glass vials and cartridges acceptable for manufactureinclude Kimbel Glass Co., West Co., Bonder Glas GMBH and Form a Vitrum.The biological and chemical properties of the PPF polypeptide may bestabilized by formulation and lyophilization in a Wheaton Type I-33borosilicate serum vial to a final concentration of 0.1 mg/ml and 10mg/ml of the PPF polypeptide in the presence of 5% mannitol, and 0.02%Tween 80. In order to permit introduction of a needle from a hypodermicsyringe into a multiple-dose vial and provide for resealing as soon asthe needle is withdrawn, the open end of each vial may be sealed with arubber stopper closure held in place by an aluminum band. Stoppers forglass vials, such as, West 4416/50, 4416/50 (Teflon faced) and 4406/40,Abbott 5139 or any equivalent stopper can be used as the closure forpharmaceutical for injection. These stoppers are compatible with thepeptide as well as the other components of the formulation. Theinventors have also discovered that these stoppers pass the stopperintegrity test when tested using patient use patterns, e.g., the stoppercan withstand at least about 100 injections. In some embodiments, thepeptide can be lyophilized in to vials, syringes or cartridges forsubsequent reconstitution. Liquid formulations of the present inventioncan be filled into one or two chambered cartridges, or one or twochamber syringes.

In some embodiments, the manufacturing process for liquid formulationsinvolves compounding, sterile filtration and filling steps. In someembodiments, the compounding procedure involves dissolution ofingredients in a specific order (preservative followed bystabilizer/tonicity agents, buffers and peptide) or dissolving at thesame time.

The United States Pharmacopeia (USP) states that anti-microbial agentsin bacteriostatic or fungistatic concentrations must be added topreparations contained in multiple dose containers. They must be presentin adequate concentration at the time of use to prevent themultiplication of microorganisms inadvertently introduced into thepreparation while withdrawing a portion of the contents with ahypodermic needle and syringe, or using other invasive means fordelivery, such as pen injectors. Antimicrobial agents should beevaluated to ensure compatibility with all other components of theformula, and their activity should be evaluated in the total formula toensure that a particular agent that is effective in one formulation isnot ineffective in another. It is not uncommon to find that a particularantimicrobial agent will be effective in one formulation but noteffective in another formulation.

In a particular embodiment of the present invention, a pharmaceuticalformulation of the present invention may contain a range ofconcentrations of PPF compounds, e.g., between about 0.01% to about 98%w/w, or between about 1 to about 98% w/w, or between 80% and 90% w/w, orbetween about 0.01% to about 50% w/w, or between about 10% to about 25%w/w. A sufficient amount of water for injection may be used to obtainthe desired concentration of solution. The pharmaceutical formulationsdescribed herein may be lyophilized. An exemplary formulation can be 1mg/mL PPF compound in 10 mM sodium acetate buffer solution, pH 4.2,containing 9.3% sucrose as an osmolality modifier.

Tonicifying agents such as sodium chloride, as well as other knownexcipients, may be present, if desired. If such excipients are present,it may be preferable to maintain the overall tonicity of the PPFpolypeptide. An excipient may be included in the presently describedformulations at various concentrations. For example, an excipient may beincluded in the concentration range from about 0.02% to about 20% w/w,between about 0.02% and 0.5% w/w, about 0.02% to about 10% w/w, or about1% to about 20% w/w. In addition, similar to the present formulationsthemselves, an excipient may be included in solid (including powdered),liquid, semi-solid or gel form.

It is possible that other ingredients may be present in theformulations. Such additional ingredients may include, e.g., wettingagents, emulsifiers, oils, antioxidants, bulking agents, tonicitymodifiers, chelating agents, metal ions, oleaginous vehicles, proteins(e.g., human serum albumin, gelatin or proteins) and a zwitterion (e.g.,an amino acid such as betaine, taurine, arginine, glycine, lysine andhistidine). Additionally, polymer solutions, or mixtures with polymersprovide the opportunity for controlled release of the peptide. Suchadditional ingredients, of course, should not adversely affect theoverall stability of the formulation of the present invention.

Generally, a therapeutically or prophylactically effective amount of thepresent PPF polypeptides will be determined by the age, weight, andcondition or severity of the diseases or metabolic conditions ordisorders of the recipient. See, e.g., Remington's PharmaceuticalSciences 697-773. See also Wang and Hanson, Parenteral Formulations ofProteins and Peptides: Stability and Stabilizers. Journal of ParenteralScience and Technology, Technical Report No. 10, Supp. 42:2S (1988).Typically, a dosage of between about 0.001 μg/kg body weight/day toabout 1000 μg/kg body weight/day, may be used, but more or less, as askilled practitioner will recognize, may be used. Dosing may be one,two, three, four or more times daily, or less frequently, such as once aweek, once a month, or once a quarter, depending on the formulation, andmay be in conjunction with other compositions as described herein. Itshould be noted that the present invention is not limited to the dosagesrecited herein.

Appropriate dosages may be ascertained through the use of establishedassays for determining level of metabolic conditions or disorders inconjunction with relevant dose-response data. The final dosage regimenwill be determined by the attending physician, considering factors thatmodify the action of drugs, e.g., the drug's specific activity, severityof the damage and the responsiveness of the patient, the age, condition,body weight, sex and diet of the patient, the severity of any infection,time of administration and other clinical factors. As studies areconducted, further information will emerge regarding appropriate dosagelevels and duration of treatment for specific diseases and conditions.

In some embodiments, an effective dose will typically be in the range ofabout 0.5 μg to about 5 mg/day, about 10 μg to about 2 mg/day, about 100μg to about 1 mg/day, or about 5 μg to about 500 μg/day, administered ina single or divided doses of two, three, four or more administration.Accordingly, exemplary doses can be derived from the total amount ofdrug to be given a day and the number doses administered a day.Exemplary doses can range from about 0.125 μg/dose (0.5 μg given fourtimes a day) to about 5 mg/dose (5 mg given once a day). Other dosagescan be between about 0.01 to about 250 μg/kg/dose. The exact dose to beadministered may be determined by one of skill in the art and isdependent upon the potency of the particular compound, as well as uponthe age, weight and condition of the individual. Administration shouldbegin whenever the suppression of nutrient availability, food intake,weight, blood glucose or plasma lipid lowering is desired, for example,at the first sign of symptoms or shortly after diagnosis of obesity,diabetes mellitus, or insulin-resistance syndrome. Administration may beby any route, e.g., injection, subcutaneous or intramuscular, oral,nasal, transdermal, etc. Dosages for certain routes, for example oraladministration, may be increased to account for decreasedbioavailability, for example, by about 5-100 fold.

In some embodiments, where the pharmaceutical formulation is to beadministered parenterally, the composition is formulation so as todeliver a dose of PPF polypeptide ranging from 0.1 μg/kg to 100 mg/kgbody weight/day. In some embodiments, the doses range from 1 μg/kg toabout 50 mg/kg body weight/day. Exemplary daily amounts may be in therange of a lower limit of 2, 5, 10, 20, 40, 60 or 80 to an upper limitof 80 100, 150, 200, or 250. Parenteral administration may be carriedout with an initial bolus followed by continuous infusion to maintaintherapeutic circulating levels of drug product. Those of ordinary skillin the art will readily optimize effective dosages and administrationregimens as determined by good medical practice and the clinicalcondition of the individual patient.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the routes of administration. The optimal pharmaceuticalformulation will be determined by one of skill in the art depending onthe route of administration and the desired dosage. See, e.g.,Remington's Pharmaceutical Sciences, supra, pages 1435-1712. Suchformulations may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein, as well as thepharmacokinetic data observed in animals or human clinical trials.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention may be useful in fields of humanmedicine and veterinary medicine. Thus the subject to be treated may bea mammal. In some embodiments, the mammal is a human or other animal.For veterinary purposes, subjects include for example, farm animalsincluding cows, sheep, pigs, horses and goats, companion animals such asdogs and cats, exotic and/or zoo animals, laboratory animals includingmice, rats, rabbits, guinea pigs and hamsters; and poultry such aschickens, turkeys, ducks and geese.

In addition, the present invention contemplates a kit comprising a PPFpolypeptide of the invention, components suitable for preparing said PPFpolypeptide of the invention for pharmaceutical application, andinstructions for using said PPF polypeptide and components forpharmaceutical application.

To assist in understanding the present invention, the following Examplesare included. The experiments relating to this invention should not, ofcourse, be construed as specifically limiting the invention and suchvariations of the invention, now known or later developed, which wouldbe within the purview of one skilled in the art are considered to fallwithin the scope of the invention as described herein and hereinafterclaimed.

EXAMPLES

The present invention is described in more detail with reference to thefollowing non-limiting examples, which are offered to more fullyillustrate the invention, but are not to be construed as limiting thescope thereof. The examples illustrate the preparation of the presentPPF polypeptides, and the testing of these PPF polypeptides of theinvention in vitro and/or in vivo. Those of skill in the art willunderstand that the techniques described in these examples constitutebest modes of practice and represent techniques described by theinventors to function well in the practice of the invention. However, itshould be appreciated that those of skill in the art should, in light ofthe present disclosure, understand that many changes can be made in thespecific methods that are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Preparation of PPF Polypeptides

Peptides of the invention may be assembled on a Symphony peptidesynthesizer (Protein Technologies, Inc.) using Rink amide resin(Novabiochem) with a loading of 0.43-0.49 mmol/g at 0.050-0.100 mmol.Fmoc amino acid (5.0 eq, 0.250-0.500 mmol) residues are dissolved at aconcentration of 0.10 M in 1-methyl-2-pyrrolidinone (NMP). Otherreagents((O-Benzotriazole-N,N,N′,N′-tetraethyl-uronium-hexafluoro-phosphate(HBTU), 1-hydroxybenzotriazole hydrate (HOBt) andN,N-Diisopropylethylamine (DIEA)) are prepared as 0.55 Mdimethylformamide solutions. The Fmoc protected amino acids are thencoupled to the resin-bound amino acid using. HBTU (2.0 eq, 0.100-0.200mmol), 1-hydroxybenzotriazole hydrate (1.8 eq. 0.090-0.18 mmol),N,N-diisopropylethylamine (2.4 eq, 0.120-0.240 mmol) for 2 hours.Following the last amino acid coupling, the peptide is deprotected using20% (v/v) piperidine in dimethylformamide (DMF) for 1 hour. Once peptidesequence is complete, the Symphony peptide synthesizer is programmed tocleave the peptide from the resin. Trifluoroacetic acid (TFA) cleavageof the peptide from resin is carried out using 93% TFA, 3% phenol, 3%water and 1% triisopropylsilane for 1 hour. The cleaved peptide isprecipitated using tert-butyl methyl ether, pelleted by centrifugationand lyophilized. The pellet is re-dissolved in water (10-15 mL),filtered and purified via reverse phase HPLC using a C18 column and anacetonitrile/water gradient containing 0.1% TFA. The resulting peptidesare purified to homogeneity by reverse phase HPLC and the purity isconfirmed by LC/MS.

A general procedure for N-capping the peptides of the invention withfatty acids and Acyl functionalities (e.g., octanoic and stearic acids,and isocaproyl and isobutyloxycarbonyl modifications) is as follows:peptide on Rink amide resin (0.1 mmol) is suspended in NMP (5 mL). In aseparate vial, HBTU (0.3 mmol), HOBt (0.3 mmol) is dissolved in DMF (5mL) followed by the addition of DIEA (0.6 mmol). This solution is addedto the resin and this suspension is shaken for 2 hours. The solvent isfiltered and washed thoroughly with NMP (5 mL×4) and CH₂Cl₂ (20 mL),dried and is subjected to TFA cleavage for 1 hr. The yield of thedesired peptide is about 40 mg after cleavage and purification.N-Carbamate derivatives (isobutyloxy, isopropyloxy, n-butyloxy, ethoxy)were obtained by coupling the corresponding carbonyl chlorides andpeptides on Rink amide resin using DIEA, DMAP and dry CH2Cl2.

A general procedure for incorporating fatty acids on the epsilon aminogroup of a lysine is as follows: the modifications are carried out insolution on a free epsilon-amino group of a lysine of a purified peptidein the presence of the fatty acid and activating agent (HBTU/HOBt) inDMF. The resulting derivatives are purified by reverse phase HPLC andthe purity is confirmed by LC/MS.

PEG modification may be carried out in solution on a free epsilon-aminogroup of lysine or a terminal amino group of a purified peptide usingcommercially available activated PEG esters. The resulting PEGylatedderivatives are purified to homogeneity by reverse phase HPLC and thepurity is confirmed by LC/MS and MALDI-MS.

Intramolecular disulphide bond formation may be performed on freecysteines using iodine/acetic acid as oxidizing agent.

The PPF polypeptides of the invention may be tested in a variety ofbiological assays in vitro, including Y-receptor binding assays usingbinding assay methodologies generally known to those skilled in the art,or in vivo, using food intake, body weight, and body composition assaysusing methodologies generally known to those skilled in the art.Exemplary assays include those described below.

Assays for Signaling by G_(i)/G₀-Coupled Receptors:

Without intending to be limited by theory, G-protein coupled receptor(GPCR) signaling mediated by heterotrimeric G-proteins can becategorized into signaling classes based upon an alpha-subunitcomposition. G_(s), G_(q) and G_(i)/G₀ proteins mediate intracellularsignaling through activation of signaling pathways leading to distinctphysiological endpoints. Activation of G_(s) and G_(i)/G₀-coupledreceptors leads to stimulation or inhibition of adenylate cyclase,respectively, while activation of G_(q)-coupled receptors results instimulation of phospholipase C (PLC) and an increase in intracellularcalcium concentration. Measuring a reduction in cAMP resulting fromactivation of G_(i)/G₀-coupled receptors can be technically difficult,whereas measuring a G_(q)-coupled increase in intracellular calcium isrelatively easier. Thus, assays have been developed for assessing theactivity of G_(i)/G₀-coupled receptors using cells co-transfected withpromiscuous G-alpha subunits to redirect G_(i)/G₀ signaling through PLCand employing reporter genes or calcium sensitive fluorophores are knownin the art. These assays may be used to assess the ability of PPFpolypeptides to act as agonists or antagonists at Y-receptors. See, forexample, Stables, et al., (1997) Anal. Biochem. 252(1): 115-26.

NPY Y1 Receptor Binding Assay:

Membranes are prepared from confluent cultures of SK-N-MC cells thatendogenously expresses the neuropeptide Y1 receptors. Membranes areincubated with 60 pM [¹²⁵I]-human Peptide YY (2200 Ci/mmol, PerkinElmerLife Sciences), and with unlabeled PPF polypeptide for 60 minutes atambient temperature in a 96 well polystyrene plate. The well contentsare then harvested onto a 96 well glass fiber plate using a Perkin Elmerplate harvester. Dried glass fiber plates are combined with scintillantand counted on a Perkin Elmer scintillation counter.

NPY Y2 Receptor Binding Assay:

Membranes are prepared from confluent cultures of SK-N-BE cells thatendogenously expresses the neuropeptide Y2 receptors. Membranes areincubated with 30 pM [¹²⁵I]-human Peptide YY (2200 Ci/mmol. PerkinElmerLife Sciences), and with unlabeled PPF polypeptide for 60 minutes atambient temperature in a 96 well polystyrene plate. The well contentsare then harvested onto a 96 well glass fiber plate using a Perkin Elmerplate harvester. Dried glass fiber plates are combined with scintillantand counted on a Perkin Elmer scintillation counter.

NPY Y4 Receptor Binding Assay:

CHO-K1 cells are transiently transfected with cDNA encoding neuropeptideY4 gene, and then forty-eight hours later membranes are prepared fromconfluent cell cultures. Membranes are incubated with 18 pM [¹²⁵I]-humanPancreatic Polypeptide (2200 Ci/mmol, PerkinElmer Life Sciences), andwith unlabeled PPF polypeptide for 60 minutes at ambient temperature ina 96 well polystyrene plate. The well contents are then harvested onto a96 well glass fiber plate using a Perkin Elmer plate harvester. Driedglass fiber plates are combined with scintillant and counted on a PerkinElmer scintillation counter.

NPY Y5 Receptor Binding Assay:

CHO-K1 cells are transiently transfected with cDNA encoding neuropeptideY5 gene, and then forty-eight hours later membranes are prepared fromconfluent cell cultures. Membranes are incubated with 44 pM [¹²³I]-humanPeptide YY (2200 Ci/mmol, PerkinElmer Life Sciences), and with unlabeledPPF polypeptide for 60 minutes at ambient temperature in a 96 wellpolystyrene plate. The well contents are then harvested onto a 96 wellglass fiber plate using a Perkin Elmer plate harvester. Dried glassfiber plates are combined with scintillant and counted on a Perkin Elmerscintillation counter.

Table 2 demonstrates certain PPF polypeptides of the invention and theiractivity in various Y-receptor binding assays such as those describedabove.

TABLE 2 SEQ ID NO: Y1RBA (nM) Y2RBA (nM) Y4 RBA (nM) Y5RBA (nM) 1 101000 0.034 1.6 2 0.2 0.058 4.5 0.31 3 6.2 0.041 54 0.85 4 0.48 0.24 390.43 5 >1000 229 >1000 0.59 6 0.42 0.19 0.84 0.19 7 1000 21 1000 1000 81000 12 1000 1000 9 0.61 0.085 51 0.47 10 1.3 0.023 107 0.49 11 2.60.059 96 0.41 12 1.7 0.14 16 0.31 13 3.2 0.42 169 0.54 14 1000 1.6 10006.8 15 1.6 0.026 52 0.33 16 4.1 0.048 29 0.15 17 11 0.037 104 0.36 180.32 0.031 19 0.32 19 5.4 0.036 117 0.73 20 2.9 0.04 93 0.42 21 24 0.31182 3.3 22 12 0.1 75 7.4 23 13 0.2 54 3.2 26 4.4 0.04 120 0.42 27 7 0.18104 1.3 28 0.55 0.032 9.2 0.23 29 14 0.46 178 0.95 50 0.86 0.15 14 0.651 0.68 0.14 7.7 0.56 52 2.7 0.19 21 0.93 53 2.2 0.084 7.4 0.64 89 4.70.11 38 0.99 90 15 0.46 50 7.3 91 9.2 0.35 99 1.9 92 9.8 0.36 107 5 938.6 0.28 99 5.6 94 1.8 0.048 27 0.54 95 8.2 0.67 101 7.3 96 7.4 0.29 566.6 97 8.6 0.19 54 2.9 98 4.4 0.099 49 2.1 99 3.5 0.065 43 0.99 100 5.90.28 70 4 101 8.6 0.18 65 3.4 102 7.8 0.09 58 1.8 103 1.8 0.038 22 0.66104 4.6 0.053 27 0.89 105 4.4 0.3 68 3.3 106 5.4 0.081 37 0.92 107 110.27 70 5.1 108 8.8 0.12 51 2.1 109 9.5 0.73 74 34 110 20 0.81 97 8.7111 17 0.41 71 10 112 5.6 0.33 76 6.3 113 6.8 0.1 37 1.2 114 71 0.25 11914 115 34 6.2 193 55 116 8.9 0.23 40 10 117 7.3 0.21 74 5.8 118 88 0.97180 31 119 158 1.1 92 47 120 17 1.5 44 27 121 14 0.19 51 14 122 36 0.468 2.4 123 45 9.2 66 1.7 124 >1000 86 >1000 56 125 28 9.1 129 8.4 126 2434 88 2.4 127 >1000 >1000 >1000 >1000 128 >1000 113 >1000 >1000 130 4.50.25 46 5.2 131 6.8 0.28 80 2.9 132 17 0.56 113 7 133 1000 8.3 1000 138135 293 43 1000 1000 136 88 0.081 863 1.8 138 7.9 0.43 165 5 139 301 201000 354 140 1000 380 1000 1000 142 6.2 0.12 61 1.2 143 3.8 0.19 56 2.3144 4.5 0.39 52 4.6 145 5.4 0.12 47.5 1.5 146 8.7 0.19 73 2.3 147 5.10.092 48 1.7 148 5 0.1 50 1.8 150 276 11 1000 118 151 7.6 0.25 115 2.1152 3.7 0.24 3.9 0.82 153 8.4 0.28 135 2.9 155 7.6 0.24 108 2.3 156 7.30.35 147 3.3 157 5.8 0.11 63 1.6 158 6.1 0.11 66 2.1 160 6.3 0.56 71 2.9162 11 0.47 86 2.8 165 4.8 0.072 59 1.3 171 33 0.53 97 10 172 22 3.3 599.1 173 14 0.99 52 7.8 174 11 0.35 64 80 175 20 0.72 >1000 >1000 176 7.60.84 120 8.5 177 5.8 0.34 46 11 178 7.7 0.29 38 17 179 30 5.4 33 208 1804.3 0.11 49 3.9 181 6.3 0.41 46 2.4 182 4.4 0.21 65 5.8 183 4.7 0.071 609.2 184 26 0.14 54 42 185 3 0.13 38 3.8 186 0.85 0.11 29 2.8 187 1000 621000 128 188 1000 102 1000 968 189 1000 57 1000 202 190 1000 24 1000 578193 308 78 331 180 194 32 1.5 89 15 195 15 1.7 146 5.7 196 1000 612 10001000 197 1000 46 611 1000 198 10 0.7 88 9.9 199 38 4.1 143 58 200 106 7426 74 201 27 2.2 99 29 202 36 148 23 80 203 33 4.4 108 78 204 47 1.1223 37 205 44 1.5 172 18 206 66 15 204 45 207 180 0.69 1000 114 208 22893 407 568 211 3.7 0.24 50 5.4 212 2.9 0.046 59 0.8 225 6.7 0.15 79 1.8226 3 0.059 35 0.57 227 1 0.032 38 0.11 228 4.1 0.1 61 1.1 229 8.2 0.2357 2.7 230 3.4 0.1 45 1.2 231 5.6 0.37 55 9.4 235 8.7 0.65 77 12 236 6.50.24 62 4.6 237 2.1 0.11 35 2.8 239 0.18 0.092 18 0.27 240 2.4 0.059 890.58 241 4 0.15 61 0.88 242 2.7 0.13 71 1 243 18 0.74 124 7.2 244 11 1.588 7.5 245 0.19 0.077 16 0.35 246 3.9 0.11 119 0.7 247 0.38 0.12 25 0.76248 0.48 0.12 24 0.44 249 0.36 0.11 21 0.34 250 2.2 0.075 73 0.51 2510.42 0.12 28 0.52 252 2.1 0.074 52 0.64 253 1.3 0.041 34 0.29 254 2.30.051 85 0.56 255 5.7 0.26 208 2 256 1.7 0.039 395 0.48 258 0.39 0.12 220.89 260 0.42 0.16 22 0.74 261 2.9 0.11 71 1 262 1.7 0.087 61 0.91 2633.2 0.1 141 1.2 264 1.8 0.22 98 0.48 265 7.3 1.1 272 11 266 2 0.13 1931.7 267 0.25 0.1 9.5 0.32 268 0.31 0.14 21 0.57 269 3.8 0.084 77 0.74270 3.3 0.13 97 1.4 271 0.51 0.094 4.2 0.25 272 0.26 0.1 12 0.27 2730.32 0.18 21 0.89 274 4.9 0.42 181 1.5 275 0.59 0.099 81 1.5 276 0.680.3 8.3 1.3 277 3.4 0.16 150 2.5 278 3.6 0.078 138 1.4 279 6.4 1.2 20012 280 2.1 0.38 108 1.6 281 2.8 0.1 117 0.67 282 0.55 0.04 18 0.15 28330 3.4 87 10.6 284 1.1 0.071 47 0.56 285 0.67 0.18 16 0.54 286 0.65 0.110.75 0.3 287 5.2 0.16 10 1.2 288 1.8 0.35 11 1.1 289 48 0.83 290 1870.51 291 186 201 9.5 0.71 292 1.4 0.17 0.77 0.32 293 0.82 0.18 0.87 0.48294 0.94 0.17 0.98 0.51 295 1 0.18 1 0.63 296 2.7 0.76 2.9 2.1 297 3.60.32 4 1.8 298 5.5 1.2 3.4 3.9 299 11 3.2 16 7.5 300 83 16 311 78 301 263.7 70 28 302 5.1 0.68 93 2.9 303 6 0.5 7.1 3.3 304 0.51 0.14 0.48 0.28306 0.6 0.16 1.2 0.27 307 0.53 0.13 0.73 0.47 308 1 0.56 2.1 1.4 309 3.378 5.6 1.5 310 29 454 27 5.1 311 16 0.49 51 1.8 312 70 0.42 91 3.4 3139.2 0.57 151 2.6 314 8.2 0.67 202 2.5 315 9.2 2.1 467 5.6 316 7.1 0.6352 1.1 317 4.3 0.097 16 0.69 318 100 1.3 84 1.9 319 35 1.04 77 1.2 32077 3.1 243 13 321 12 3.7 57 5.6 332 13 0.54 38 1 333 4.8 0.54 37 0.87334 21 0.45 101 2.4 335 34 0.72 109 3.6 338 8.1 0.68 46 1.1 341 1.8 0.1511 0.3 342 15 0.62 84 1.4 343 12 0.38 69 1.3 347 35 18 740 51 436 4 0.0736 1.4 437 5.1 0.45 371 2.1 438 1.5 0.079 167 1.2 439 0.93 0.05 176 0.47440 1.6 0.1 100 1.2 441 4.8 0.65 224 7 442 1.6 0.11 214 1.3 443 474 113914 592 444 6.6 0.36 97 3.8 445 9.1 0.56 269 6.3 446 13 1 141 6.6 4478.3 0.5 206 25 448 6.6 0.1 61 1.1 449 3.6 0.068 78 3.1 450 1000 0.511000 11 451 7.8 0.89 71 18 452 7 0.34 62 3 453 0.7 0.084 17 0.82 454 4.40.27 278 6.1 455 4.5 0.81 146 5.3 456 8.5 1.1 246 10 458 10 0.47 593 3.6459 79 0.48 100 6.2 460 1.4 0.08 115 0.59 461 6.5 0.59 303 3.3 462 8.20.91 356 10 463 23 4 361 19 467 2.7 0.17 158 1.5 468 5.7 0.74 283 5.6469 3.4 0.48 508 5.1 470 6.8 0.78 585 12 471 2.6 0.18 178 3.1 472 11 1.5368 20 473 7.3 0.95 212 5.4 474 0.68 0.3 8.3 1.3 475 26 6.7 358 21 47627 7.4 53 15 477 265 4.4 164 18 478 1000 31 273 17 479 1.8 0.357 74 3.5480 7.7 2.2 211 18

Example 2 PPF Polypeptides Suppress Food Intake in Food Intake Assay

Female NIH/Swiss mice (8-24 weeks old) are group housed with a 12:12hour light:dark cycle with lights on at 0600. Water and a standardpelleted mouse chow diet are available ad libitum, except as noted.Animals are fasted starting at approximately 1500 hrs, one day prior toexperiment. The morning of the experiment, animals are divided intoexperimental groups. In a typical study, n=4 cages with 3 mice/cage.

At time=0 min, all animals are given an intraperitoneal injection ofvehicle or compound in an amount ranging from about 10 nmol/kg to 100nmol/kg, and immediately given a pre-weighed amount (10-15 g) of thestandard chow. Food is removed and weighed at 30, 60, and 120 min todetermine the amount of food consumed (Morley, Flood et al., Am. J.Physiol. 267: R178-R184, 1994). Food intake is calculated by subtractingthe weight of the food remaining at the 30, 60, 120, 180 and/or 240minute time points, for example, from the weight of the food providedinitially at time=0. Significant treatment effects were identified byANOVA (p<0.05). Where a significant difference exists, test means arecompared to the control mean using Dunnett's test (Prism v. 2.01,GraphPad Software Inc. San Diego, Calif.).

FIGS. 1-4 show the ability of several PPF polypeptides of the inventionto reduce cumulative food intake in the food intake assay describedabove. Furthermore, FIG. 40 shows that acute administration of PPFpolypeptide compound 4883 was found to be more effective than PYY(3-36)in reducing food intake in the NIH/Swiss mouse and HSD rat models.

Example 3 PPF Polypeptides Decrease Body Weight Gain in High Fat Fed(Diet-Induced-Obesity, or DIO) C57BL/6 Mice and High Fat-Fed HSD Rats

Mice:

Male C57BL/6 mice (4 weeks old at start of study) are fed high fat (HF,58% of dietary kcal as fat) or low fat (LF, 11% of dietary kcal as fat)chow. After 4 weeks on chow, each mouse is implanted with an osmoticpump (Alzet #2002) that subcutaneously delivers a predetermined dose ofPPF polypeptide continuously for two weeks. Body weight and food intakeare measured weekly (Surwit et al., Metabolism—Clinical andExperimental, 44: 645-51, 1995). Effects of the test compound areexpressed as the mean+/−sd of % body weight change (i.e., % change fromstarting weight) of at least 14 mice per treatment group (p<0.05 ANOVA,Dunnett's test, Prism v. 2.01, GraphPad Software Inc., San Diego,Calif.).

Rats:

The night before treatment, male Sprague-Dawley® rats (averageweight=415) consuming a high fat diet (45% kCal from fat) were assignedto two treatment groups based on equal 24 hr food intake. On test night,each animal received a single IP injection of Vehicle (10% DMSO) orCompound (1 mg/kg) just prior to lights off (1800 h), and were thenplaced individually into a DietPro automated feeding cage. Each cagecontains a food hopper resting on a scale connected to a computer, and awater bottle. Hourly food intake (in grams) is recorded for thefollowing 24 hours. Animals received injections for six consecutivenights. Body weights were recorded nightly.

FIGS. 5-6 demonstrate the ability of several PPF polypeptides of theinvention to decrease body weight gain in the DIO mouse assay describedabove. FIG. 7 demonstrates that once daily injections resulted in asignificant reduction in body weight gain on several nights (P<0.05) inhigh fat-fed rats. For example. FIG. 8 demonstrates that a PPFpolypeptide of the invention exhibits greater efficacy than PYY(3-36) inboth the food intake assay and the DIO mouse assay. For example, FIG. 42demonstrates the effects of another PPF polypeptide of the invention onfeeding pattern, and shows that PPF polypeptide compound 4883 reducesfood intake on nights 3 and 5, significantly reduces weight over sevendays, and reduces total food consumption for six days.

Example 4 PPF Polypeptides Reduce Blood Pressure

Male Harlan Sprague Dawley (HSD) rats housed at 22.8±0.8° C. in a 12:12hour light:dark cycle were used to study the effects of PPF Polypeptideson the circulatory system through the use of telemetry. The experimentswere performed during the light cycle. Telemetry allows for real-timehemodynamic readings including arterial blood pressure, heart rate andarterial dP/dt, via an implanted radio transmitter in conscious,non-anesthetized, unrestrained rats. In the present Example, rats wereinjected with either vehicle, 10 nmol/kg PYY, 10 nmol/kg PYY(3-36) or 10nmol/kg of several PPF polypeptides by remote intravenous dosing. Remoteintravenous dosing was achieved through in-dwelling vascular accessports (Access Technologies (Skokie, Ill.). The port is secured to theunderlying muscle just below the skin between the scapulae. The catheterresides in the jugular vein. Data were collected for up to 60 minutesfollowing injection.

As shown in FIGS. 9A-B, the effect of compound 4676 to increase meanarterial pressure (MAP) are similar to those of PYY(3-36). FIGS. 9C-Dshow that while the effects of compound 4247 to increase mean arterialpressure and decrease heart rate are similar to those of PYY(1-36),those effects are blunted with compound 4560.

FIG. 46 demonstrates that PPF polypeptide compound 4753 also decreasesheart rate as compared to PYY(3-36), while its effect on MAP iscomparable to that of PYY(3-36). FIG. 47 demonstrates that the effectsof PPF polypeptide compound 4883 on heart rate and MAP are comparable tothose of PYY(3-36).

Example 5 Antisecretory Effects of PYY and PYY Agonists

Gastric Acid Secretion

Male Harlan Sprague Dawley rats were housed at 22.8±0.8° C. in a 12:12hour light:dark cycle. The experiments were performed during the lightcycle. Animals, fed rat chow (Teklad LM 485, Madison, Wis.), were fastedfor approximately 20 hours before experimentation. They were given freeaccess to water until the start of the experiment.

The rats (age 11-16 weeks, body mass 291-365 g) were surgically fittedwith gastric fistulae custom made by David Osborne, Department ofBiology, UCLA. Overnight fasted rats were weighed and their gastricfistulae were uncapped and attached to flexible Tygon tubing (⅜× 1/16)into which was fitted a piece of PE205 tubing that would extend into thestomach. Saline was injected through the narrower PE205 tubing and theeffluent collected from the Tygon tubing. To ensure proper flow throughthe fistulae and an empty stomach, the stomach was flushed several timeswith ˜5 ml of room temperature saline solution until flow was easy andthe effluent was clean. Gastric acid secretion was measured at 10 minintervals by injecting 5 mL of saline (pH 7.0) followed by 3 ml of airand collecting the effluent. Three ml of each gastric aspirate weretitrated to 7.0 with 0.01 N sodium hydroxide using a pH meter (Beckmanmodel number PHI34 Fullerton, Calif.). The amount of base required foreach titration, corrected to the total volume collected, was used tocalculate the moles of acid in each sample.

After a baseline sample was collected, and the recovered volumerecorded, the animal was given a subcutaneous injection of 125 μg/kgpentagastrin (Sigma, lot #40K0616) and then 10 min. gastric sampling wascontinued for a further 2 hours. Forty minutes after pentagastrininjection, when a stable plateau of gastric acid secretion was typicallyobserved, the rats were given a subcutaneous injection of (PYY(3-36)) ata dose per animal of 1, 3, 10, 100 μg or saline, (3.45, 10.34, 34.5,344.8 μg/kg, respectively, in a rat weighing 290 grams) (n=3, 2, 4, 4, 6respectively).

As shown in FIG. 10, gastric acid output was expressed as % ofpentagastrin-stimulated secretion, calculated as the average of timepoints 20, 30, and 40 minutes after injection of pentagastrin. Inresponse to pentagastrin, gastric acid secretion increased 6.8-fold froma basal rate of 9.3±5.8 μmol/10 min to 62.8±3.8 μmol/10 min 40 min afterinjection (grand means: P<0.01). PYY(3-36) injected 40 min afterpentagastrin dose-dependently and significantly inhibited gastric acidproduction. With doses of 10 μg (34.5 μg/kg) and 100 μg (344.8 μg/kg),PYY(3-36), acid secretion was reduced by 74.7±7.2% and 84.7±9.7%,respectively (P<0.05 and P<0.01; t-test, 20 minutes after PYY(3-36)injection) (see t=60 min in FIGS. 11-17). The dose response forPYY(3-36) inhibition of pentagastrin-stimulated acid secretion is shownin FIG. 11. The ED₅₀ for the antacid effect of PYY(3-36) was 11.31μg/kg±0.054 log units.

Gastric Emptying

To determine the effects of PYY [3-36] on gastric emptying, conscious,non-fasted male Harlan Sprague Dawley rats were randomly divided intothree overall treatment groups: 1) for animals designated “APx,”vacuum-aspiration lesions were made to the area postrema; 2) for theanimals designated “Sham,” to control for effects of the surgery,sham-operations were performed in which the cranial region wassurgically opened, but no lesion was made to the brain and 3) unoperatedcontrol animals, designated “Control.” were not subjected to surgery.For each of the three overall treatment groups, animals were thendivided into dosage groups, in which they were administered eithersaline only, or bolus doses of 3, 30, 90, or 300 μg/kg PYY(3-36)dissolved in saline. Experiments were performed at least two weeks postsurgery (weight 426±8 g) and again three weeks later (weight 544±9 g).All rats were housed at 22.7° C. in a 12:12 h light:dark cycle(experiments performed during light cycle) and were fed and watered adlibitum (diet LM-485 Teklad, Madison, Wis., USA).

PYY(3-36) dissolved in saline was administered as a 0.1 ml subcutaneousbolus 5 min before gavage of 5 μCi D-[3-³H]-glucose (lot #3165036Dupont, Wilmington, Del., USA) in 1 ml water. The vehicle or differentdoses of PYY was given subcutaneously after animals had been given anoral liquid meal.

There were 15 Treatment Groups:

(1) Control saline n = 4 (2) Control 3 μg/kg n = 3 (3) Control 30 μg/kgn = 4 (4) Control 90 μg/kg n = 5 (5) Control 300 μg/kg n = 5 (6) Shamsaline n = 5 (7) Sham 3 μg/kg n = 2 (8) Sham 30 μg/kg n = 4 (9) Sham 90μg/kg n = 3 (10) Sham 300 μg/kg n = 5 (11) APx saline n = 5 (12) APx 3μg/kg n = 3 (13) APx 30 μg/kg n = 3 (14) APx 90 μg/kg n = 3 (15) APx 300μg/kg n = 5

Blood was collected from anesthetized tails of the rats at −15, 0, 5,15, 30, 60 and 90 min after gavage for measurements and the plasmaseparated to measure the plasma glucose-derived tritium (CPM per 10 μlcounted in β-counter). The appearance of tritium in plasma haspreviously been shown to reflect gastric emptying. The integratedtritium appearance in plasma was calculated using the trapezoidal methodas the increment above the levels before the tritium gavage (thearea-under-the-curve (AUC) for 30 minutes).

In unoperated control rats, PYY(3-36) dose-dependently inhibited labelappearance, (10.5±1.5, 7.26±1.52 and 3.20±1.21 cpm/μL·min for 30 μg/kg,90 μg/kg, 300 μg/kg PYY(3-36), respectively; P<0.0001 ANOVA; see FIGS.12 and 13). In sham-operated rats, 30 μg/kg (n=4) and 90 μg PYY(3-36)injections (n=3) also delayed appearance of label compared tosaline-injected controls (n=5) in dose dependent manner (11.89±3.23,9.88±2.45, 18.94±3.23 cpm/μL/min, respectively; P<0.05 ANOVA; see FIGS.14 and 15). Maximal effect of PYY in sham-operated animals was lesscompared to intact unoperated control rats with ED₅₀ also lower than inunoperated control animals (decreases from 43.77 to 10.20 μg/kg PYY[3-36]. In APx rats, gastric emptying was slowed compared to that insham-operated or unoperated control rats (9.38±3.25 cpm/μL/min; P<0.05,0.05, see FIGS. 16 and 17), but was not altered by administration ofPYY(3-36). Regression analysis confirmed absence of dose dependency.

Results showed that PYY(3-36) potently regulates the rate of gastricemptying in normal Sprague Dawley rats. A dose-dependent inhibition ofgastric emptying was observed following the injection of PYY(3-36) (30,90 and 300 μg/kg). AP-lesioned animals had a tendency to delay gastricemptying compared to unoperated control and sham-operated rats (n.s.).PYY(3-36) administration had no additional effect on gastric emptyingrate in the AP-lesioned animals.

FIG. 45 demonstrates that administration of PPF polypeptide compound4883 is more potent than PYY(3-36) in inhibiting gastric emptying.

Gallbladder Emptying

In the processes of normal digestion, gastric emptying rates andgallbladder emptying rates may be coordinated. Circulating PYY has beenreported to suppress the cephalic phase of postprandial gallbladderemptying, but not meal stimulated maximum emptying. It was alsohypothesized that the effect of PYY on gallbladder emptying is mediatedby vagal-dependent rather than cholecystokinin-dependent pathways(Hoentjen, F, et al., Scand. J. Gastroenterol. 2001 36(10):1086-91). Todetermine the effect of PYY [3-36] on gallbladder emptying, eight weekold, male NIH Swiss mice were housed at 22.8±0.8° in a 12:12 light:darkcycle, and allowed ad libitum access to a standard rodent diet (TekladLM 7012, Madison, Wis.) and water. The mice were food deprived for 3hours prior to experimentation. At t=0, PYY(3-36), CCK-8 or saline wasinjected subcutaneously in conscious mice. Thirty min later, mice wereeuthanized by cervical dislocation, a midline laparotomy was performedand the gallbladder was excised and weighed.

Treatment Groups:

Group A: saline 100 μl subcutaneously at t=−0, n=14.

Group B: PYY(3-36) 1 μg/kg subcutaneously at t=0, n=6.

Group C: PYY(3-36) 10 μg/kg subcutaneously at t=0, n=10.

Group D: PYY(3-36) 100 μg/kg subcutaneously at t=0, n=8.

Group E: CCK-8 1 μg/kg subcutaneously at t=0, n=3.

Group F: CCK-8 10 μg/kg subcutaneously at t=0, n=3.

Group G: PYY(3-36) 10 μg/kg+CCK-8 μg/kg subcutaneously at t=0, n=4.

Group H: PYY(3-36) 10 μg/kg+CCK-8 10 μg/kg subcutaneously at t=0, n=4.

The results are shown in FIGS. 18 and 19. PYY(3-36) dose dependentlyinhibited basal gallbladder emptying with an ED₅₀ of 9.94 μg/kg±0.24 logunits. The highest dose (Group D) increased gallbladder weight by 168%over that observed in saline injected controls (Group A) (P<0.005).PYY(3-36) did not affect CCK-8 stimulated gallbladder emptying. The dataindicate that PYY(3-36) inhibits gallbladder emptying viaCCK-independent pathways. Gallbladder emptying in response to exogenousCCK was not affected by PYY(3-36). A similar result was obtained withPYY[1-36] in conscious dogs; a 400 ng/kg bolus+800 pmol/kg/h infusiondid not inhibit CCK-8-stimulated gallbladder contraction.

It is possible that the effects of PYY(3-36) on gallbladder emptying aremediated by vagal-cholinergic pathways. This idea is supported byfindings that specific peptide YY (PYY) binding sites have recently beenautoradiographically identified in the area postrema, nucleus of thesolitary tract, and dorsal motor nucleus regions (collectively referredto as the dorsal vagal complex (DVC)) in rats. These medullary brainstem regions are responsible for vagovagal reflex control ofgastrointestinal functions, including motility and secretion. PYY(3-36)inhibits other digestive functions that are mediated byvagal-cholinergic mechanisms, such as gastric emptying.

Example 6 Gastroprotective Effects of PYY and PYY Agonists

Male Harlan Sprague Dawley rats were housed at 22.8±0.8° in a 12:12light:dark cycle, and allowed ad libitum access to a standard rodentdiet (Teklad LM 485, Madison, Wis.) and water. The rats, 200-220 gm,were fasted for approximately 20 hours prior to experimentation.

At t=−30, PYY(3-36) or saline was injected s.c. At t=0, a 1 ml gavage ofabsolute ethanol (ethyl alcohol-200 proof dehydrated alcohol, U.S.P.punctilious) or saline was administered. At t=30, the rats wereanesthetized with 5% isoflorane. A midline laparotomy incision was made.The stomach was exposed and ligated at the pyloric and lower esophagealsphincters. The stomach was excised, opened along the lesser curvatureand everted to expose the mucosa. The mucosa was gently rinsed withsaline and assessed for damage (ulcerations, dilated blood vessels,sloughing off of the mucosal lining) by observers blinded to thetreatment. Mucosal damage was scored between 0 (no damage) and 5 (100%of stomach covered by hyperemia and ulceration).

Treatment Groups:

Group A: saline 100 μl s.c. at t=−30, gavage 1 ml H2O at t=0, n=4.

Group B: saline 100 μl s.c. at t=−30, gavage 1 ml absolute ethanol att=0, n=6.

Group C: PYY(3-36) 1 μg/kg at t=−30, gavage 1 ml absolute ethanol att=0, n=5.

Group D: PYY(3-36) 10 μg/kg at t=−30, gavage 1 ml absolute ethanol att=0, n=4.

Group E: PYY(3-36) 100 μg/kg at t=−30, gavage 1 ml absolute ethanol att=0, n=5.

Group F: PYY(3-36) 300 μg/kg at t=−30, gavage 1 ml absolute ethanol att=0, n=5.

PYY(3-36) dose dependently reduced the injury score by 27.4±6.4,29.3±11.6 and 53.7±7.9% (n=4, 5, 5, p<0.05 ANOVA) after injection of 10,100, and 300 μg/kg of PYY(3-36), respectively (FIG. 20). PYY [3-36]showed a gastroprotective effect, in rats. Endogenously circulating PYY[3-36] may play a physiologic role in controlling gastric acid secretionand protecting the gastric mucosa.

Examples 7-10 Effects of PPF Polypeptides on Food Intake, Body WeightGain, Metabolic Rate and Body Composition

In rodents, weight reduction following administration of PYY(3-36) maybe attributable to decreased food consumption or other processesimpacting energy balance (including energy expenditure, tissue-levelfuel partitioning, and/or gut nutrient uptake). The effects ofcontinuous subcutaneous infusion of PYY(3-36) (1 mg/kg/day, up to 7days) on metabolic rate, fat combustion, and/or fecal energy loss indiet-induced obese (DIO) mice were examined.

Animal Care and Housing

Examples 7-10 utilized a diet-induced obese (DIO) mouse model formetabolic disease. Prior to the treatment period, male C57BL/6J micewere fed a high-fat diet (#D12331, 58% of calories from fat; ResearchDiets, Inc.) for 6 weeks beginning at 4 weeks of age. During the study,the mice remained on this high-fat diet in powdered form throughout thetreatment period unless otherwise noted. Water was provided ad libitumthroughout the study. Animals were housed under a 12 hr:12 hr light-darkcycle at 21-23° C., and allowed ad libitum access to food pre- andpost-treatment. In some embodiments, eight wk-old male NIH Swiss(non-obese) mice (HarlanTeklad, Indianapolis, Ind., USA) fed a standardchow diet (Teklad #LM7012, Madison, Wis.) were used in gallbladderemptying experiments. Where noted, one group of similarly-aged nonobesemice were fed a low-fat diet (#D12329, 11% of calories from fat) forpurposes of comparing metabolic parameters to DIO groups.

Peptide Source

In some embodiments, the trifluoroacetic acid salt of human PYY(3-36)(>98% purity) was synthesized using standard methods (Peptisyntha,Torrance, Calif.), and its identity confirmed using mass spectroscopy.

Experimental Designs, Blood and Tissue Collection for Body CompositionAnalyses.

Studies of metabolic parameters [Study A], nutrient uptake by the gut[Studies B and C], food intake and body composition [Studies B and C]used mice that were housed singly for 1 week prior to treatment.Throughout the experiments, food intake and body weight were monitoreddaily. During the treatment period, vehicle (50% dimethylsulfoxide inwater) and PYY(3-36) (1 mg/kg/day) were administered by continuoussubcutaneous (s.c.) infusion using Alzet® osmotic pumps (Durect Corp.,Cupertino, Calif.; Models 1003D, 2001, & 2004 for 3, 7, & 28 daystudies, respectively) placed in the intrascapular region underisoflurane anesthesia. At the end of each study, animals were sacrificedafter a 2-4 hr fast by isoflurane overdose. Blood was collected intoNa-heparin-flushed syringes by cardiac puncture, and plasma wasimmediately frozen. In some studies (Studies B and C), body compositionwas determined using dual-energy X-ray absorptiometry (DEXA; PixiMus, GELunar). Bilateral epididymal fat pads and intrascapular brown adiposetissue (BAT) were dissected and weights determined. Excised liversamples were placed in RNALater (Ambion, Austin, Tex.), and stored at−20° C.

Indirect Calorimetry [Study A].

DIO mice were acclimated to indirect calorimetry cages for 4 days priorto measuring post-treatment metabolic rate and RQ (Oxymax; softwareversion 2.52; Columbus Instruments, Columbus, Ohio). The within-animalCV % during the 2-day pre-treatment baseline, averaged 4.6±0.8% &4.0±0.8% for light & dark cycle energy expenditure, respectively,indicating adequate acclimation. Following osmotic pump implantation(vehicle controls, n=13: PYY(3-36) at 1 mg/kg/day, n=12), calorimetricmeasurements were made continuously over 7 days. Heat production wascalculated by the instrument software (based on Lusk, G., (1928) TheElements of the Science of Nutrition, 4th Ed., W.B. Saunders Company,Philadelphia) and is reported relative to body mass measured on eachtreatment day.

Fecal Energy Analysis [Studies B and C].

Mice were acclimated to metabolic cages (Diuresis Cages; Nalge NuncInt'l Corp., Rochester, N.Y.; Study B), or to standard cages with raisedwire mesh flooring [Study C], and to powdered high-fat chow for 4 daysprior to treatment. In Study B, fecal energy content was determinedusing bomb calorimetry (Covance Labs; Madison, Wis.). To collectsufficient material, a pooling strategy was used for each mouse: pooledsamples from individuals' 2 day baseline period, early treatment period(Days 1, 2, 3) and late treatment period (Days 5, 6, 7) were compared.In Study C, fecal energy content was determined in samples collectedover the final 24 hr from cage bottoms lined with absorbent paper.

Long-Term Effects of PYY(3-36) on Body Weight in DIO Mice.

Vehicle (n=18) or PYY(3-36) (n=−24; 300 μg/kg/day, the estimated ED₅₀for weight change in a prior study in this model (Pittner, et al.,(2004) Int. J. Obes. Relat. Metab. Disord. 28: 963-71) were administeredto DIO mice by Alzet s.c. osmotic pumps. At 28 days, pumps werereplaced: controls continued to receive vehicle, and half of thePYY(3-36) group (n=12) continued to receive the peptide. The other halfof the PYY(3-36) group (n=12), which had received PYY(3-36) for theinitial treatment period, received new pumps containing vehicle to testthe effect of peptide withdrawal. Mice were fed pelleted high-fat diet,and body weights and food intake were recorded weekly.

Gallbladder Emptying in Mice.

Non-obese mice in the postabsorptive state (3 hr fasted) were injecteds.c. with saline (n=14) or PYY(3-36) at 1, 10, or 100 μg/kg (n=6, 11, 8,respectively). Animals were sacrificed by cervical dislocation at 30min. post-injection, and gallbladders were removed and weighed as ameasure of gallbladder emptying rate.

Biochemical Assays.

Plasma β-hydroxybutyrate (Cat. #2440, STANBIO Laboratory, Boerne, Tex.),glycerol (Cat. #TR0100, Sigma, St. Louis, Mo.), and non-esterified fattyacids (NEFA C, Cat. #994-75409, Wako Chemicals, Richmond, Va.) weremeasured using standard colorimetric assays. Total PYY immunoreactivityin plasma was determined by Linco Diagnostic Services (St. Louis, Mo.)using a human PYY RIA displaying <0.1% cross-reactivity to mouse or ratPYY(3-36), and averaged 39.3 ng/ml (˜10 nM) in mice treated with 1mg/kg/day PYY(3-36). Ex vivo lipolysis (glycerol release over 1 hr) wasmeasured in non-obese female mouse retroperitoneal fat pad preparationsusing the method of Heffernan (Heffernan, et al., (2000) Am. J. Physiol.Endocrinol. Metab. 279: E501-7). Fat pads were incubated with PYY(3-36)at concentrations ranging from the upper physiologic to pharmacologicplasma levels (0.05, 0.5, & 10 nM) Values were compared to basal ratesfrom untreated adipose tissue.

Statistical comparisons between control and treated animals over time(Examples 7 and 8) were made using a two-way analysis of variance(ANOVA) determining the effects of time, treatment, and time×treatmentinteraction (Prism v. 4.01, GraphPad Software, San Diego, Calif.).Differences between control and treated groups were analyzed by t-tests.Differences were considered statistically significant at p<0.05. In someembodiments, differences between treatment group means for parametersdetermined over time were analyzed using a repeated measures analysis ofvariance; post-hoc tests within timepoints were tested for simpleeffects using pooled standard error (SPSS version 13.0, Chicago, Ill.).Two-group comparisons were carried out using a Student's t-test, anddose-response data were evaluated using a one-way ANOVA and Tukey'scomparison. Data are presented as mean±SEM, with p<0.05 considered to bestatistically significant.

Gene Expression Analyses.

RNA for gene expression analyses was isolated from a subset of tissuesper manufacturer's instructions (RiboPure kit #1924; Ambion). One-stepquantitative real-time RT-PCR analysis was used to measure mRNAabundance (ABI 7900HT; Applied Biosystems, Inc., Foster City, Calif.).The 50 μl reaction conditions were: 2.5 μL Assay-on-Demand® primer/probemix, 1× Master Mix, 1× Multiscribe/RNAse inhibitor mix, and 50 ng RNA.RT-PCR conditions were: 48° C. 30 min., 95° C. 10 min., then 40 cycles(95° C. 15 sec/60° C. 1 min.). For each gene, cycle numbers werecorrected for loading variation by simultaneously assaying 18S RNAabundance using a commercially-available primer/probe set (ABI). Therelative abundance of mRNAs corresponding to the following genes weredetermined using ABI Assay-on-Demand® primer/probe sets: liver-typecarnitine palmitoyltransferase 1 (L-CPT1 or CPT1a; Mm00550438_ml),acetyl-CoA carboxylase 1 (ACC1; Mm01304257_ml), ACC2 (Mm01204677_ml),mitochondrial hydroxymethylglutaryl-CoA synthase (HMGCS2;Mm00550050_ml), malonyl-CoA decarboxylase (MCD or MLYCD; Mm01245664_ml),and uncoupling protein 1 (UCP1; Mm00494069_ml). The results of thesegene expression analyses are shown in Table 3 below. mRNA abundance isexpressed as fold-difference vs. vehicle-treated control values within agiven treatment time. *P<0.05 vs. Vehicle.

TABLE 3 Treatment time 3 Days 7 Days Genes and Vehicle PYY(3-36) VehiclePYY(3-36) groups (n = 8) (n = 9) (n = 8) (n = 7) L-CPT1 1.00 ± 0.08 0.93± 0.08 1.00 ± 0.05 1.17 ± 0.15 ACC1 1.00 ± 0.11 1.02 ± 0.11 1.00 ± 0.10 1.36 ± 0.13* ACC2 1.00 ± 0.13 0.86 ± 0.11 1.00 ± 0.09  1.47 ± 0.17* MCD1.00 ± 0.09 0.77 ± 0.09 1.00 ± 0.10 0.89 ± 0.10 HMGCS2 1.00 ± 0.11  0.68± 0.10* 1.00 ± 0.05 1.04 ± 0.08

Example 7

Singly-housed DIO mice were implanted with subcutaneous (SC)intrascapular osmotic pumps to deliver either vehicle (50%dimethylsulfoxide [DMSO] in water) n=13 or synthetic human PYY(3-36)n=12. The pumps of the latter group were set to deliver 1000 μg/kg/d ofPYY(3-36) for 7 days.

Body weights and food intake were measured over regular intervalsthroughout the study periods. Respiratory quotient (RQ, defined as CO2production. O2 consumption) and metabolic rate were determined usingwhole-animal indirect calorimetry (Oxymax, Columbus Instruments,Columbus, Ohio).

The mice were euthanized by isoflurane overdose, and an index ofadiposity (bilateral epididymal fat pad weight) was measured.

Example 8

This experiment essentially repeated the study described in Example 7,with n=9 per group (vehicle and PYY(3-36)). However, prior todetermination of epididymal weight, body composition (lean mass, fatmass) for each mouse was analyzed using a Dual Energy X-rayAbsorptiometry (DEXA) instrument per manufacturer's instructions (LunarPiximus, GE Imaging System).

FIGS. 21A and 21B show the change in body weight as a percentage ofbaseline for DIO mice continuously administered vehicle or PYY(3-36)(1000 μg/kg/d) for 7 days. FIG. 21A shows the results of Example 7 andFIG. 21B shows the results of Example 8, and significance is denoted as*p<0.05, **p<0.01, ***p<0.001 vs. controls.

FIGS. 22A and 22B show the change in food intake as a percentage ofbaseline for DIO mice continuously administered vehicle or PYY(3-36)(1000 μg/kg/d) for 7 days. FIG. 22A shows the results of Example 7 andFIG. 22B shows the results of Example 8, and significance is denoted as*p<0.05, **p<0.01, ***p<0.001 vs. controls. There appears to be a trendfor reduced food intake at Day 3 (

indicates p=0.06) in FIG. 22A and Day 5 (

indicates p=0.1) in FIG. 22B.

The respiratory quotient (RQ) of the mice in Example 7 was measured andcompared. The RQ in PYY(3-36)-administered DIO mice was reduced duringseveral dark cycle periods, and was lower during the light cyclethroughout the study period. An RQ value near 0.70 is indicative ofreliance upon fat catabolism to meet the energy requirements of theanimal; thus, the relatively lower RQ in PYY(3-36)-administered animalsis consistent with increased fat utilization for energy vs. control mice(*p<0.05, **p<0.01, ***p<0.001 vs. controls). This effect is especiallypersistent during the light cycle when animals are in a postabsorptivestate (reduced food intake relative to the dark cycle) (see FIGS. 23Aand 23B). These results indicate that PYY(3-36) has properties whichdrive fat combustion to meet caloric requirements which may lead to apreferential loss of fat over protein.

Moreover, the reduced RQ observed with PYY(3-36) administration in DIOmice relative to vehicle-administered control is indicative of improvedfat utilization for energy at the tissue- and cell-level (increasedfatty acid β-oxidation). The majority of metabolic rate and RQ isinfluenced by metabolism in non-adipose tissues, such as liver andskeletal muscle. It follows then that PYY, PYY(3-36) and agoniststhereof could be therapeutically useful in situations in which improvedfatty acid β-oxidation in non-adipose tissues is desirable, withmaintenance of lean body mass. Examples of such conditions include, butare not limited to, nonalcoholic steatohepatitis and lipodystrophy. Amore specific example may be in the treatment of AIDS patients who aretaking protease inhibitors. These patients may suffer from lipodystrophy(irregular fat distribution) that tends to increase central, truncalgirth while at the same time decrease fat in the arms and legs. Thetreatment goal would be the reduction of central fat and an increase inperipheral muscle mass.

FIGS. 24A, 24B, 25A and 25B, for example, show evidence that PYY(3-36)and its agonists have the property of preferentially inducing the lossof fat over the loss of lean body tissue. Epididymal fat pads of Example7 and Example 8 mice were weighed, and the reduced epididymal fat padweights of the PYY(3-36)-administered mice over vehicle-administeredmice, as shown in FIGS. 24A and 24B, Examples 7 and 8 respectively,indicate reduced adiposity in DIO mice administered PYY(3-36),(**p<0.01. ***p<0.001 vs. controls). In addition, reduced adiposity ofPYY(3-36)-administered mice is supported by lower whole-animal fat massdetermination by DEXA of Example 8 mice (FIG. 25A; **p<0.01 vs.controls). Of particular interest from the DEXA results is that despitesignificant weight loss (FIG. 21B) and fat loss (FIG. 24B and FIG. 25A),lean body mass was maintained in PYY(3-36)-administered mice, and didnot differ much from those of vehicle-administered mice (FIG. 25B).

Example 9

In this experiment, the dose-effect of PYY(3-36) was studied and over alonger period than previous experiments. DIO mice were implanted with SCintrascapular osmotic pumps to deliver either vehicle (saline) orPYY(3-36). The pumps of the latter group were set to deliver a range ofdoses up to 1000 μg/kg/d for 28 days. Body weights and food intake weremeasured over regular intervals throughout the study periods.

Mice were housed two per cage. Sample sizes for body weight and foodintake in this experiment were n=20, n=14, and n=12 for High-fatcontrols, Low-fat comparator group, and High-fat-fed PYY(3-36) groups,respectively. For body composition measures, sample sizes were n=18,n=14, and n=12 for High-fat controls, Low-fat comparator group, andHigh-fat-fed PYY(3-36) groups, respectively.

FIG. 26 shows the change in body weight as a percentage of initial bodyweight of vehicle-administered mice fed low-fat chow,vehicle-administered DIO mice fed high-fat chow, andPYY(3-36)-administered DIO mice fed high-fat chow. Increasing doses ofPYY(3-36) show an increasing effect on body weight (*p<0.05 vs.controls).

FIG. 27 shows the weekly food intake of the mice during four weeks ofthe study. The group of low-fat fed mice and the PYY(3-36) (1000μg/kg/d) high-fat fed DIO mice consistently consumed significantly lessfood than the high-fat fed DIO mice controls during the four weeks ofthe study.

FIGS. 28A and 28B show that while fat mass was lower in low-fat fed miceand PYY(3-36) (1000 μg/kg/d) high-fat fed DIO mice (*p<0.01; ***p<0.001vs. vehicle-administered high-fat fed DIO mice controls), low-fat fedmice had significantly less protein mass whereas PYY(3-36) (1000μg/kg/d)-administered DIO mice did not have significantly less proteinmass than the high-fat fed controls. Whole carcass body composition wasdetermined by proximate analysis using standard methods (CovanceLaboratories, Madison, Wis.).

Example 10

In another study similar to those carried out in Examples 7 and 8,singly-housed DIO mice were implanted with subcutaneous (SC)intrascapular osmotic pumps to deliver either vehicle (50%dimethylsulfoxide [DMSO] in water) n=10 or synthetic human PYY(3-36)n=10. The pumps of the latter group were set to deliver 1000 μg/kg/d ofPYY(3-36) for 3 days.

FIGS. 29A and 29B show the change in body weight and food intake,respectively, as a percentage of baseline for DIO mice continuouslyadministered vehicle or PYY(3-36) (1000 μg/kg/d) for 3 days. FIGS. 29Aand 29B show significant reduction in body weight and food intake in theDIO mice administered PYY(3-36) over the course of the treatment period(*p<0.05, **p<0.01, ***p<0.001 vs. controls).

FIG. 30 shows significantly less adiposity in DIO mice administeredPYY(3-36) over controls as indicated by lower epididymal fat pad weight(**p<0.01 vs. control). FIG. 31A shows significantly less adiposity inDIO mice administered PYY(3-36) over controls as indicated by lowerwhole-animal fat mass determination by DEXA, as described in Example 8(**p<0.01 vs. control). While DIO mice administered PYY(3-36)significantly lost body weight and reduced food intake and had lessadiposity over controls, FIG. 31B shows that the lean body mass of thePYY(3-36)-administered mice did not differ significantly from controls.

FIGS. 32A and 32 B show the effects of administration of vehicle orPYY(3-36) on metabolic rate during the light cycle (top panel) and darkcycle (bottom panel) in DIO mice. Symbols: closed circles,vehicle-treated controls; open diamonds, PYY(3-36)-treated (1 mg/kg/day,continuous subcutaneous infusion). Day 0 represents the baseline(pre-treatment) mean value (25.2±0.3 kcal/kg/hr & 30.8±0.3 kcal/kg/hrfor the light and dark cycle, respectively).

FIG. 33 shows the acute effects of i.p. PYY(3-36) injection ongallbladder emptying in non-obese mice. Gallbladder weights measured at30 min. post-injection are depicted, with a higher weight reflective ofreduced basal gallbladder emptying rate. ***p<0.001, significantlydifferent vs. saline-treated controls.

FIGS. 34A and 34B show the effect of prolonged PYY(3-36) administrationand withdrawal in DIO mice on body weight and food intake, respectively.DIO mice were treated with PYY(3-36) (300 μg/kg/day) or vehicle for upto 56 days; PYY(3-36) was withdrawn from some animals after 28 days andreplaced with vehicle. Symbols: closed circles, vehicle-treatedcontrols; open diamonds, PYY(3-36)-treated; closed diamonds, PYY(3-36)days 0-28 followed by vehicle days 28-56. The initial (pre-treatment)mean weight was 24.7±1.6 g. Body weight differed significantly inPYY(3-36)-treated mice at all timepoints (p≦50.001 vs vehicle control);body weight in mice withdrawn from PYY(3-36) did not differ fromcontrols by day 35 or thereafter.

Overall, continuous subcutaneous infusion of PYY(3-36) (1 mg/kg/day, upto 7 days) was observed to increase metabolic rate, fat combustion,and/or fecal energy loss in diet-induced obese (DIO) mice. PYY(3-36)transiently reduced food intake (e.g., 25-43% lower at Day 2 relative topre-treatment baseline) and decreased body weight (e.g., 9-10% reducedat Day 2 vs. baseline). The effect on body weight was durable,persisting throughout a 56-day study. Withdrawal of PYY(3-36) after 28days of treatment was associated with transiently increased food intake,and regain of weight to the control level. Mass-specific metabolic rate(kcal/kg/hr) did not differ from controls. Light cycle RQ was reduced byPYY(3-36) throughout the study (averaging 0.730±0.006 vs. 0.750±0.009 incontrols; p<0.001). Dark cycle respiratory quotient (RQ) was transientlydecreased in PYY(3-36)-treated mice (e.g., Day 2, 0.747±0.008 vs.0.786±0.004 in controls; p<0.001). Epididymal fat pad weight inPYY(3-36)-treated mice was decreased by approximately 50%. Fat padlipolysis ex vivo was not stimulated by PYY(3-36), nor were therechanges in the expression of hepatic genes relevant to lipid metabolism.PYY(3-36) decreased basal gallbladder emptying in non-obese mice;however, fecal energy density (kcal/100 g) did not change sufficientlyto impact energy balance.

In some embodiments, in-bred male DIO prone rats were obtained fromCharles Rivers Laboratories. These rats were developed from a line ofCrl:CD® (SD)BR rats that are prone to become obese on a diet relativelyhigh in fat and energy. These animals rapidly gain weight and body fatresulting in a hyper-triglyceridemic, -leptinemic and -insulinemicstate. They were housed individually in shoebox cages at 22° C. in a12:12-hour light dark cycle. Rats were maintained ad-libitum on amoderately high fat diet (32% kcal from fat; Research Diets D1226B) for6 weeks prior to drug treatment. At the end of the fattening periodtheir body weights were ˜500 g. Chronic administration of test compoundswas by subcutaneous osmotic pump. Indirect calorimetry was performed at1 week. Plasma analytes were analyzed on day 14 after an overnight fast.Analyses of food intake, body weight, body weight gain, bodycomposition, metabolic rate, RQ, EE, gastric acid secretion, gastricemptying, gallbladder emptying, and statistical comparisons wereperformed as described above.

FIG. 35 depicts an example of the dose-dependent decrease in cumulativefood intake and percent change in body weight observed uponadministration of PYY(3-36) in inbred DIO prone rats at day 14. Based onthese data, a dose of 500 μg/kg/day of PYY(3-36) was chosen forexperiments exploring the effects of combining PYY(3-36) with otheragents marketed for the treatment of obesity, appetite control oraltering body composition, such as, for example, but not limited to, anamylin, amylin agonist or amylin analog agonist, salmon calcitonin, acholecystokinin (CCK) or CCK agonist, a leptin (OB protein) or leptinagonist, an exendin or exendin analog agonist, a glucagon-like peptide-1(GLP-1), GLP-1 agonist or GLP-1 analog agonist, CCK, CCK agonists,calcitonin, calcitonin agonists, small molecule cannabinoid CB1 receptorantagonists, rimonabant, 11 beta-hydroxysteroid dehydrogenase-1inhibitors, phentermine, or sibutramine. In some embodiments, a dose of500 μg/kg/day of PYY(3-36) was combined with a dose of 100 μg/kg/day ofamylin. In some embodiments, a dose of 200 μg/kg/day of PYY(3-36) wascombined with a dose of 100 μg/kg/day of amylin.

For example, FIG. 36 depicts exemplary effects of administering 200μg/kg/day of PYY(3-36) with and without co-administration of 100μg/kg/day of amylin on body weight as well as on fasting plasmaparameters in DIO prone rats. Co-administration of PYY(3-36) was foundto have an additive effect in reducing body weight. A glucose-loweringeffect of administration of PYY(3-36) alone was also observed.Furthermore, co-administration of amylin and PYY(3-36) reducedtriglyceride levels in an additive manner, without reducing HDLcholesterol levels. The additive effect on weight loss observed uponco-administration of PYY(3-36) and amylin was accompanied by a reductionin respiratory quotient (RQ) without a significant reduction in energyexpenditure (EE) in these DIO prone rats (see FIG. 37).

The additive effect on weight loss observed upon co-administration ofPYY(3-36) and amylin was also accompanied by significant reduction infat tissue mass, without concomitant reduction in protein mass relativeto vehicle (see FIG. 38). Thus, the combination of PYY(3-36) and amylinappears to be effective in altering body composition via lean-sparingbody fat reduction.

The stability of several PPF polypeptides in human plasma was tested andcompared to the plasma stability of PYY(3-36). To assess in vitrodegradation, each of the PPF polypeptides or PYY(3-36) was incubated inhuman plasma at 37° C. for three hours, and aliquots were removed atspecified time-points and analyzed for peptide concentration. Thepeptide concentration was determined by comparison with a standardcurve, and the degradation rates were determined by calculating theslope of the change in concentration over time. A comparison between thedegradation rate of a PPF polypeptide and that of PYY(3-36) is shown inFIG. 39. In this example, PPF polypeptide compound 4883 has enhancedplasma stability as compared to PYY(3-36). FIG. 54 compares thecalculated rates of degradation of several other PPF polypeptides tothat of PYY(3-36). It was observed that compounds 4676, 4247 and 4753have enhanced plasma stability in this assay as compared to PYY(3-36),whereas compound 4757 is less stable than PYY(3-36).

In mouse and rat DIO models, chronic administration of PPF polypeptidecompound 4883 was found to have increased efficacy in reducing bodyweight as compared to PYY(3-36) (see FIG. 41).

In some embodiments, a PPF polypeptide can preferentially lower plasmatriglycerides, without changing other plasma analytes, such as HDLcholesterol, glucose or HbA1C levels. In some embodiments, a PPFpolypeptide can lower plasma triglycerides and amylase levels, withoutchanging other plasma analytes, such as HDL cholesterol, glucose orHbA1C levels. In some embodiments, the reduction in plasma triglyceridelevels is greater than the reduction in cholesterol levels. In someembodiments, plasma triglyceride levels are lowered and LDL cholesterollevels are lowered to a lesser extent. FIG. 43 demonstrates that chronicadministration of PPF polypeptide compound 4883 in DIO rats over 28 daysalters body composition by reducing fat tissue mass without changinglean tissue mass, and FIG. 44 depicts the preferential lowering oftriglyceride levels by administration of PPF polypeptide compound 4883.

In some embodiments, the PPF polypeptide and another agent, such asamylin, are administered via the same subcutaneous pump. In someembodiments, the PPF polypeptide and another agent, such as amylin, areadministered through separate subcutaneous pumps. FIG. 48 depictsexemplary effects of administering 500 μg/kg/day of PYY(3-36) or a PPFpolypeptide with and without co-administration of 100 μg/kg/day ofamylin on body weight in DIO prone rats. Co-administration of 500μg/kg/day of PYY(3-36) and 100 μg/kg/day of amylin was found to have anadditive effect in reducing body weight. FIGS. 48 and 49 show that PPFpolypeptide compounds 4883 and 4917 are more potent than PYY(3-36) inreducing body weight. FIGS. 48 and 49 also show that co-administrationof 500 μg/kg/day of PPF polypeptide compound 4883 or compound 4917 plus100 μg/kg/day of amylin have a correspondingly greater additive effectin reducing body weight as compared to the additive effect of PYY(3-36)plus amylin. The additive effect on weight loss observed uponco-administration of PPF polypeptide compound 4883 plus amylin wasaccompanied by a reduction in respiratory quotient (RQ) and energyexpenditure (EE) in these DIO prone rats (see FIG. 53).

The additive effect on reduction of body weight in DIO prone ratsobserved upon co-administration of amylin plus PPF polypeptide compound4883 or 4917 was accompanied by a significant reduction in fat tissuemass, without a significant loss of lean tissue (see FIGS. 50 and 51).The co-administration of compound 4883 and amylin appears to have asynergistic effect on reducing body weight (FIG. 51). Overall these datademonstrate that co-administration of amylin plus a PPF polypeptide isan effective means of altering body composition via lean-sparing bodyfat reduction.

FIG. 52 shows that PPF polypeptide compound 4917, with or withoutco-administration of amylin, is more effective than PYY(3-36) atlowering fasting insulin levels in DIO rats.

Certain PPF polypeptides are shown in Table 4 below, although otherpolypeptides are envisioned. The following abbreviations may be used:hK=homolysine, hR=homoarginine, hS=homoserine, hP=homoproline,G(oct)=octylglycine, Aib=2-aminoisobutyric acid, Cit=citruline,Dap=diaminopropionic acid, Sar=sarcosine.

TABLE 4 ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 A P L E P V YP G D N A T P E Q M A 2 Y P I K P E A P G E D A S P E E L N 3 I K P E AP G E D A S P E E L N 4 Y P S K P D N P G E D A P A E D M A 5 A P L E PV Y P G D N A T P E Q M A 6 A P L E P V Y P G D N A T P E Q M A 7 Y P PK P E S P G E N A T P E E L A 8 P K P E S P G E N A T P E E L A 9 A P PK P E H P G D D A P A E D V A 10 P K P E H P G D D A P A E D V A 11 P KP E N P G E D A P P E E L A 12 A Y P P K P E S P G D A A S P E E I A 13M P P K P D N P S S D A S P E E L S 14 P K P D N P S S D A S P E E L S15 P K P D N P G D N A S P E Q M A 16 P K P E N P G D N A S P E E M A 17T K P E N P G N D A S P Q E M A 18 Y P P K P E N P G E D A S P E E M T19 P K P E N P G E D A S P E E M T 20 I K P E A P G E D A S P E E L A 21S K P D N P G E D A P A E D M A 22 P K P E H P G D D A P A E D V N 23 PK P E H P G D D A P A E D V N 24 P K P E H P G D D A P A E D V A 25 I PE H P G D D A P A E D V A 26 P K P E H P G D D A P A E D V A 27 P K P EH P G D D A P A E D V A 28 Y P P K P E A P G E D A S P E E L A 29 A K PE N P G D N A P A E Q M A 30 FORMULA I 31 Y P I hK P E A P G E D A S P EE L N 32 Y P I K P A A P G E D A S P E E L N 33 Y P I K P E A P A E D AS P E E L N 34 Y P I K P E A P G A D A S P E E L N 35 Y P I K P E A P GE A A S P E E L N 36 Y P I K P E A P G E D A A P E E L N 37 Y P I K P EA P G E D A S A E E L N 38 Y P I K P E A P G E D A S P A E L N 39 Y P IK P E A P G E D A S P E A L N 40 Y P I K P E A P G E D A S P E E A N 41Y P I K P E A P G E D A S P E E L A 42 Y P I K P E A P G E D A S P E E LN 43 Y P I K P E A P G E D A S P E E L N 44 Y P I K P E A P G E D A S PE E L N 45 Y P I K P E A P G E D A S P E E L N 46 Y P I K P E A P G E DA S P E E L N 47 Y P I K P E A P G E D A S A E E L A 48 Y P I K P E A PG E D A S P E E L A 49 Y P I K P E A P G E D A S A E E L A 50 A P I K PE A P G E D A S P E E L N 51 F P I K P E A P G E D A S P E E L N 52 Y dAI K P E A P G E D A S P E E L N 53 Y G I K P E A P G E D A S P E E L N54 A Y P I K P E A P G E D A S P E E L 55 Y A P I K P E A P G E D A S PE E L 56 Y P A I K P E A P G E D A S P E E L 57 Y P I A K P E A P G E DA S P E E L 58 Y P I K A P E A P G E D A S P E E L 59 Y P I K P A E A PG E D A S P E E L 60 Y P I K P E A A P G E D A S P E E L 61 Y P I K P EA P A G E D A S P E E L 62 Y P I K P E A P G A E D A S P E E L 63 Y P IK P E A P G E A D A S P E E L 64 Y P I K P E A P G E D A A S P E E L 65Y P I K P E A P G E D A S A P E E L 66 Y P I K P E A P G E D A S P A E EL 67 Y P I K P E A P G E D A S P E A E L 68 Y P I K P E A P G E D A S PE E A L 69 Y P I K P E A P G E D A S P E E L A 70 Y P I K P E A P G E DA S P E E L N 71 Y P I K P E A P G E D A S P E E L N 72 Y P I K P E A PG E D A S P E E L N 73 Y P I K P E A P G E D A S P E E L N 73 Y P I K PE A P G E D A S P E E L N 74 Y P I K P E A P G E D A S P E E L N 75 Y PI K P E A P G E D A S P E E L N 76 Y P I K P E A P G E D A S P E E L N77 Y P I K P E A P G E D A S P E E L N 78 Y P I K P E A P G E D A S P EE L N 79 Y P I K P E A P G E D A S P E E L N 80 Y P I K P E A P G E D AS P E E L N 81 Y P I K P E A P G E D A S P E E L N 82 Y P I K P E A P GE D A S P E E L N 83 Y P I K P E A P G E D A S P E E L N 84 Y P I K P EA P G E D A S P E E L N 85 Y P I K P E A P G E D A S P E E L N 86 Y P IK P E A P G E D A S P E E L N 87 Y P I K P E A P G E D A S P E E L N 88FORMULA II 89 A K P E A P G E D A S P E E L N 90 I A P E A P G E D A S PE E L N 91 I G P E A P G E D A S P E E L N 92 I dA P E A P G E D A S P EE L N 93 I K A E A P G E D A S P E E L N 94 I K P A A P G E D A S P E EL N 95 I K P E dA P G E D A S P E E L N 96 I K P E A A G E D A S P E E LN 97 I K P E A P A E D A S P E E L N 98 I K P E A P G A D A S P E E L N99 I K P E A P G E A A S P E E L N 100 I K P E A P G E D dA S P E E L N101 I K P E A P G E D A A P E E L N 102 I K P E A P G E D A S A E E L N103 I K P E A P G E D A S P A E L N 104 I K P E A P G E D A S P E A L N105 I K P E A P G E D A S P E E A N 106 I K P E A P G E D A S P E E L A107 I K P E A P G E D A S P E E L N 108 I K P E A P G E D A S P E E L N109 I K P E A P G E D A S P E E L N 110 I K P E A P G E D A S P E E L N111 I K P E A P G E D A S P E E L N 112 I K P E A P G E D A S P E E L N113 I K P E A P G E D A S P E E L N 114 I K P E A P G E D A S P E E L N115 I K P E A P G E D A S P E E L N 116 I K P E A P G E D A S P E E L N117 I K P E A P G E D A S P E E L N 118 I K P E A P G E D A S P E E L N119 I K P E A P G E D A S P E E L N 120 I K P E A P G E D A S P E E L N121 I K P E A P G E D A S P E E L N 122 I K P E A P G E D A S P E E L N123 I K P E A P G E D A S P E E L N 124 I K P E A P G E D A S P E E L N125 I K P E A P G E D A S P E E L N 126 I K P E A P G E D A S P E E L N127 I K P E A P G E D A S P E E L N 128 I K P E A P G E D A S P E E L N129 S K P D N P G E D A P A E D M A 130 P K P E A P G E D A S P E E L N131 isocap P K P E A P G E D A S P E E L N 132 AC P K P E A P G E D A SP E E L N 133 I K P E A P G E D A S P E E L N 134 I K P E A P G E D A SP E E L N 135 I K P E A P G E D A S P E E L N 136 I K P E A P G E D A SP E E L N 137 I K P E A P G E D A S P E E L N 138 I K P E A P G E D A SP E E L N 139 I K P E A P G E D A S P E E L N 140 I K P E A P G E D A SP E E L N 141 I K P E A P G E D A S P E E L N 142 I K P E A P G E D A SA E E L A 143 I K P E A P G E D A S P E E L A 144 I K P E A P G E D A SA E E L A 145 I hK P E A P G E D A S P E E L N 146 I K P E A P G E D AS(Ac) P E E L N 147 I K P E A P G E D A S P E E L N 148 I K P E A P G ED A S P E E L N 149 I K P E A P G E D A S P E E L N 150 Fmoc I K(Fmoc PE A P G E D A S P E E L N SO3H SO3H) 151 iso- I K P E A P G E D A S P EE L A caproyl 152 Fmoc I K P E A P G E D A S P E E L A 153 isobutyl- I KP E A P G E D A S P E E L A oxy- carbonyl 154 isopropyl- I K P E A P G ED A S P E E L A oxy- carbonyl 155 n-butyl- I K P E A P G E D A S P E E LA oxy- carbonyl 156 ethoxy- I K P E A P G E D A S P E E L A carbonyl 157I K P E A P G E D A S P E E L S 158 I K P E A P G E D A S P E E L nV 159I hK P E A P G E D A S P E E L A 160 isocapryl I hK P E A P G E D A S PE E L A 161 I K P E A P G E D A S P E E L A 162 isocaproyl I K P E A P GE D A S P E E L A 163 I hK P E A P G E D A S P E E L A 164 isocaproyl IhK P E A P G E D A S P E E L A 165 I K P E A P G E D A S P E E M A 166 IhK P E A P G E D A S A E E L A 167 I hK P E A P G E D A S A E E L A 168L hK P E A P G E D A hS A E E L A 169 I hK P E A P G E D A S A Q E L A170 I KA P E A P G E D A S P E E L N 171 I K P E C P G E D A S P E E C N172 L E P V Y P G E D A S P E E L A 173 L E P V Y P G E D A S P E E L A174 I K P E A P G E D A S P E E L N 175 P E A P G E D A S P E E L N 176E D A S P E E L N 177 E E L N 178 179 180 Y P I K Aminocaproyl P E E L N181 Y P I K Aminocaproyl E E L N 182 Y P I K Aminocaproyl E L N 183 Y PI K Aminocaproyl 184 Y P I K Aminocaproyl 185 Y P I K P E A P G E A N186 Y P I K P E A P G E Ado N 187 Fmoc I K(PEG P E A P G E D A S P E E LN 5000) 188 Fmoc K(PEG P E A P G E D A S P E E L N 5000) 189 Fmoc I A PE A P G E D A S P E E L N 190 PEG I A P E A P G E D A S P E E L N 5000191 I K P E A P G E D A S(O- P E E L N acyl- ation w/FA 192 I K P E A PG E D A S P E E L N 193 Fmoc I A P E A P G E D A S P E E L N 194 I A P EA P G E D A S P E E L N 195 octanoic I A P E A P G E D A S P E E L Aacid 196 I A P E A P G E D A S P E E L N 197 Fmoc I A P E A P G E D A SP E E L N 198 Ac E D A S P E E L N 199 stearyl E D A S P E E L N 200octyl E D A S P E E L N 201 succinyl E D A S P E E L N 202 203 204 I A PE A P G E D A S P E K(PEG) L N 205 I A P E A P G E D A S P E K(Oct) L N206 Fmoc I A P E A P G E D A S P E K(Oct) L N 207 I K P E A P G E D A SP E E L N 208 I K P E A P G E D A S P E E L N 209 I K P E A P G E D A SP E E L N 210 I K P E A P G E D A S P E E L N 211 + I K P E A mimic E DA S P E E L N 352 A 212 + I K P E A P mimic D A S P E E L N 353 A 213 +I K P E A P G mimic A S P E E L N 354 A 214 + I K P E A P G E mimic S PE E L N 355 A 215 + I K P E A P G E D mimic P E E L N 356 A 216 + I K PE A P G E D A mimic E E L N 357 A 217 + I K P E A P G E D A S mimic E LN 358 A 218 I K P E A A Aib E D A S P E E L N 219 I K P E A P A Aib D AS P E E L N 220 I K P E A P G A Aib A S P E E L N 221 I K P E A P G E AAib S P E E L N 222 I K P E A P G E D A Aib P E E L N 223 I K P E A P GE D A A Aib E E L N 224 I K P E A P G E D A S A Aib E L N 225 I K P E AA P E D A S P E E L N 226 I K P E A P A P D A S P E E L N 227 I K P E AP G A D A S P E E L N 228 I K P E A P G E A P S P E E L N 229 I K P E AP G E D A P P E E L N 230 I K P E A P G E D A S A P E L N 231 + I K P EA mimic E D A S P E E L N 359 B 232 + I K P E A P mimic D A S P E E L N360 B 233 + I K P E A P G mimic A S P E E L N 361 B 234 + I K P E A P GE mimic S P E E L N 362 B 235 + I K P E A P G E D mimic P E E L N 363 B236 + I K P E A P G E D A mimic E E L N 364 B 237 + I K P E A P G E D AS mimic E L N 365 B 238 A P L E P V Y P G D N A T P E Q M A 239 Y P I KP E A P G E D A S P E E L A 240 I K P E A P G E D A S P E E L A 241 I KP E A P G E D A S P E E L A 242 isocap I K P E A P G E D A S P E E L A243 I P E A P G E D A S P E E L A 244 Y P I P E A P G E D A S A E E L A245 Y P I K P E A P G E D A S P E E L A 246 I K P E A P G E D A S P E EL A 247 Y P I K P E A P G E D A S A E E L A 248 Y P I K P E A P G E D AS A E E L A 249 Y P I hK P E A P G E D A S P E E L A 250 I hK P E A P GE D A S P E E L A 251 Y P I hK P E A P G E D A S P E E L A 252 I hK P EA P G E D A S P E E L A 253 P hK P E A P G E D A S P E E L A 254 P K P EA P G E D A S P E E L A 255 isocap P K P E A P G E D A S P E E L A 256 PK P E H P G E D A P A E E L A 257 P K P E A P G E D A P A E E L A 258 YP I hK P E A P G E D A S P E E L A 259 I hK P E A P G E D A S P E E L A260 Y P I hK P E A P G E D A S P E E L A 261 I hK P E A P G E D A S P EE L A 262 P hK P E A P G E D A S P E E L A 263 P K P E A P G E D A S P EE L A 264 P P K P E A P G E D A S P E E L A 265 isocap P K P E A P G E DA S P E E L A 266 P K P E H P G E D A P A E E L A 267 Y P I R P E A P GE D A S P E E L A 268 Y P I R P E A P G E D A S P E E L A 269 I R P E AP G E D A S P E E L A 270 P R P E A P G E D A S P E E L A 271 8-aminooctanoyl I R P E A P G E D A S P E E L A 272 Y P I R P E A P G E D A S PE E L A 273 Y P I K P E A P G E D A P A E E L A 274 I K P E A P G E D AP A E E L A 275 P Y P I K P E A P G E D A P A E E L A 276 8-aminooctanoyl K P E A P G E D A P A E E L A 277 P K P E A P G E D A P A E E LA 278 P K P E A P G E D A P A E E L A 279 isocap P K P E A P G E D A P AE E L A 280 isocap Y P I K P E A P G E D A S P E E L A 281 isocap I K PE A P G E D A S P E E L A 282 G(Oct) I K P E A P G E D A S P E E L A 283Fmoc-G(Oct) I K P E A P G E D A S P E E L A 284 G(Oct) K P E A P G E D AS P E E L A 285 Y P S K P D N P G E D A P A E D M A 286 A P L E P V Y PG D N A T P E Q M N 287 L E P V Y P G D N A T P E Q M N 288 A P L E P VY P G D N A T P E Q M N 289 A P L E P V Y P G D N A T P E Q M N 290 A PL E P V Y P G D N A T P E Q M N 291 A P L E P V Y P G D N A T P E Q M N292 A P L E P V Y P G D N A T P E Q M N 293 A P L E P V Y P G D N A T PE Q M N 294 A P L E P V Y P G D N A T P E Q M A 295 A P L E P V Y P G DN A T P E Q L N 296 A P L E P V Y P G D N A T P E E L N 297 A P L E P VY P G D N A S P E E L N 298 A P L E P V Y P G D D A S P E E L N 299 A PL E P V Y P G E D A S P E E L N 300 A P L E P V A P G E D A S P E E L N301 A P L E P E A P G E D A S P E E L N 302 A P L K P E A P G E D A S PE E L N 303 isocap P L E P V Y P G D N A T P E Q M N A 304 A P L E P V YP G D N A T P E Q M N 305 isocap P L E P V Y P G D N A T P E Q M N A 306A P L E P V Y P G D N A T P E Q M N 307 A P M E P V Y P G D N A T P E QM N 308 A P L E P V Y P G D N A T P E Q M N 309 Y P I K P E A P G E D AS P E E L A 310 I K P E A P G E D A S P E E L A 311 Y P I K P E A P G ED A S P E E L A 312 I K P E A P G E D A S P E E L A 313 Y P I K P E A PG E D A S P E E L A 314 Y P I K P E A P G E D A S P E E L A 315 Y P I KP E A P G E D A S P E E L A 316 Y P I K P E A P G E D A S P E E L N 317Y P I K P E A P G E D A S P E E L N 318 Y dA I K P E A P G E D A S P E EL A 319 A P I K P E A P G E D A S P E E L A 320 isocap P I K P E A P G ED A S P E E L A Y 321 Y P I K P E A P G E D A S P E E L A 322 Y P I K PE A P G E D A S P E E L A 323 Y P I K P E A P G E D A S P E E L A 324 YP I K P E A P G E D A S P E E L A 325 Y P I K P E A P G E D A S P E E LA 326 Y P I K P E A P G E D A S P E E L A 327 Y P I K P E A P G E D A SP E E L A 328 Y P I K P E A P G E D A S P E E L A 329 Y P I K P E A P GE D A S P E E L A 330 Y P I hK P E A P G E D A S P E E L A 331 isocap PI hK P E A P G E D A S P E E L A Y 332 Y P I hR P E A P G E D A S P E EL A 333 Y P I K P E A hP G E D A S P E E L A 334 Y P I K P E A Aib G E DA S P E E L A 335 Y P I K P E A G P E D A S P E E L A 336 Y P I K P E AP G E D A S hP E E L A 337 Y P I K P E A P G E D A S Aib E E L A 338 Y PI K P E A P Sar E D A S P E E L A 339 Y P I K P E A P G E D A hS P E E LA 340 Y P I K P E A P G E D A Dap P E E L A 341 Y P I K P E A P G E D AS P Q E L A 342 Y P I K P E A P G E D A S P D E L A 343 Y P I K P E A PG E D A S P E E M A 344 Y P I hK P E A P G E D A S A Q E L A 345 Y P IhK P E A P G E D A S A S E L A 346 Y P I hK P E A P G E D A S A Q E M A347 Y P I K P E A P G E D A S P E E L A 348 Formula III 349 Formula IV350 Formula V 351 hPYY C-terminal Motiff - 32-35 436 P K P E A P G E D AS P E E L N 437 isocap P K P E H P G E D A S A E E L A 438 P K P E H P GE D A S P E E L A 439 P K P E H P G E D A S A E E L A 440 P K P E A P GE D A S P E E L A 441 isocap P K P E A P G E D A S P E E L A 442 P K P EH P G E D A S A E E L A 443 I K P E A P G E D A S P E E L N 444isobutyl- P K P E A P G E D A S P E E L N oxycarbonyl 445 isocap P K P EH P G E D A S P E E L N 446 isocap P K P E A P G E D A S P E E I N 447isocap P K P E A P G E D A S P E E L N 448 P K P E A P G E D A S P E E IN 449 P K P E A P G E D A S P E E L N 450 I K P E A P G E D A S P E E LN 451 P K P E A P G E D A S P E E L N 452 Densyl I K P E A P G E D A S PE E L N 453 I K A P E A P G E D A S P E E L N 454 isocap P K P E A P G ED A S P E D L A 455 octanoyl P K P E A P G E D A S P E E L A 456isobutyl- P K P E A P G E D A S P E E L A oxycarbonyl 457 octylGly P K PE A P G E D A S P E E L A 458 isocap P K P E A P G E D A S P E E L A 459isocap P K P E A P G E D A S P E E L A 460 P K P E A P G E D A S A E E LA 461 isocap P K P E A P G E D A S A E E L A 462 isocap P K P E H P G ED A S P E E L A 463 isocap P S P E A P G E D A S P E E L A 464 isocap PK P E G P A E D A S P E E L A 465 P K P E A P G E D P S P E E L A 466isocap P K P E A P G E D P S P E E L A 467 V K P E A P G E D A S P E E LA 468 isocap V K P E A P G E D A S P E E L A 469 isocap P K P E H P G ED A P A E E L A 470 isocap P K P E H P G E D A P A E E L A 471 P K P E HP G E D A P S E E L A 472 isocap P K P E H P G E D A P S E E L A 473isocaproyl I K P E A P G E D A P A E E L A 474 8-amino octanoyl I K P EA P G E D A P A E E L A 475 BH P I K P E A P G E D A S P E E L A modi-fied- Y 476 BH P I A P E A P G E D A S P E E L A modi- fied- Y 477 BH AP E A P G E D A S P E E L A modi- fied- I 478 isocap I K-(BH P E A P G ED A S P E E L A modi- fied) 479 BH K P E A P G E D A S P E E L A modi-fied- P 480 BH A P E A P G E D A S P E E L A modi- fied- P 481 FormulaVI 482 Formula VII ID 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 3536 37 1 Q Y A A D L R R Y I N M L T R P R Y 2 R Y Y A S L R H Y L N L VT R Q R Y 3 R Y Y A S L R H Y L N L V T R Q R Y 4 R Y Y S A L R H Y I NL I T R Q R Y 5 R Y Y S A L R H Y I N L A Aib R Q R Y 6 R Y Y S A L R HY I N L I T R Q R Y 7 K Y I S A D R H Y I N L V T R Q R Y 8 K Y I S A DR H Y I N L V T R Q R Y 9 K Y Y T A L R H Y I N L I T R Q R Y 10 K Y Y TA L R H Y I N L I T R Q R Y 11 K Y Y S A L R H Y I N L I T R Q R Y 12 QY F S A L R H Y I N L V T R Q R Y 13 K Y M L A V R N Y I N L I T R Q R Y14 K Y M L A V R N Y I N L I T R Q R Y 15 R Y K A A V R H Y I N L I T RQ R Y 16 K Y F S A L R H Y I N L V T R Q R Y 17 K Y M T A L R H Y V N LI T R Q R Y 18 K Y L T A L R H Y I N L V T R Q R Y 19 K Y L T A L R H YI N L V T R Q R Y 20 K Y Y T A L R H Y I N L I T R Q R Y 21 K Y Y T A LR H Y I N L I T R Q R Y 22 R Y Y A S L R H Y L N L V T R Q R Y 23 R Y YA A L R A Y L N L V T R Q R Y 24 Q Y A A D L R R Y I N M L T R Q R Y 25R Y Y S A L R A Y I N L I T R Q R Y 26 R Y Y S A L R H Y I N L I T R Q RY 27 R Y Y S A L R A Y I N L I T R Q R Y 28 K Y Y A A L R H Y I N L V TR Q R Y 29 K Y L T A L R A Y V N L I T R Q R Y 30 31 R Y Y A S L R H Y LN L V T R Q R Y 32 R Y Y A S L R H Y L N L V T R Q R Y 33 R Y Y A S L RH Y L N L V T R Q R Y 34 R Y Y A S L R H Y L N L V T R Q R Y 35 R Y Y AS L R H Y L N L V T R Q R Y 36 R Y Y A S L R H Y L N L V T R Q R Y 37 RY Y A S L R H Y L N L V T R Q R Y 38 R Y Y A S L R H Y L N L V T R Q R Y39 R Y Y A S L R H Y L N L V T R Q R Y 40 R Y Y A S L R H Y L N L V T RQ R Y 41 R Y Y A S L R H Y L N L V T R Q R Y 42 K Y Y A S L R H Y L N LV T R Q R Y 43 R Y A A S L R H Y L N L V T R Q R Y 44 R Y Y A A L R H YL N L V T R Q R Y 45 R Y Y A S L R A Y L N L V T R Q R Y 46 R Y Y A S LR H Y L N A V T R Q R Y 47 R Y Y A S L R H Y L N L V T R Q R Y 48 R Y YA S L R A Y L N L V T R Q R Y 49 R Y Y A S L R A Y L N L V T R Q R Y 50R Y Y A S L R H Y L N L V T R Q R Y 51 R Y Y A S L R H Y L N L V T R Q RY 52 R Y Y A S L R H Y L N L V T R Q R Y 53 R Y Y A S L R H Y L N L V TR Q R Y 54 N R Y Y A S L R H Y L N L V T R Q R Y 55 N R Y Y A S L R H YL N L V T R Q R Y 56 N R Y Y A S L R H Y L N L V T R Q R Y 57 N R Y Y AS L R H Y L N L V T R Q R Y 58 N R Y Y A S L R H Y L N L V T R Q R Y 59N R Y Y A S L R H Y L N L V T R Q R Y 60 N R Y Y A S L R H Y L N L V T RQ R Y 61 N R Y Y A S L R H Y L N L V T R Q R Y 62 N R Y Y A S L R H Y LN L V T R Q R Y 63 N R Y Y A S L R H Y L N L V T R Q R Y 64 N R Y Y A SL R H Y L N L V T R Q R Y 65 N R Y Y A S L R H Y L N L V T R Q R Y 66 NR Y Y A S L R H Y L N L V T R Q R Y 67 N R Y Y A S L R H Y L N L V T R QR Y 68 N R Y Y A S L R H Y L N L V T R Q R Y 69 N R Y Y A S L R H Y L NL V T R Q R Y 70 A R Y Y A S L R H Y L N L V T R Q R Y 71 R A Y Y A S LR H Y L N L V T R Q R Y 72 R Y A Y A S L R H Y L N L V T R Q R Y 73 R YY A A S L R H Y L N L V T R Q R Y 73 R Y Y A S S L R H Y L N L V T R Q RY 74 R Y Y A S A L R H Y L N L V T R Q R Y 75 R Y Y A S L A R H Y L N LV T R Q R Y 76 R Y Y A S L R A H Y L N L V T R Q R Y 77 R Y Y A S L R HA Y L N L V T R Q R Y 78 R Y Y A S L R H Y A L N L V T R Q R Y 79 R Y YA S L R H Y L A N L V T R Q R Y 80 R Y Y A S L R H Y L N A L V T R Q R Y81 R Y Y A S L R H Y L N L A V T R Q R Y 82 R Y Y A S L R H Y L N L V AT R Q R Y 83 R Y Y A S L R H Y L N L V T A R Q R Y 84 R Y Y A S L R H YL N L V T R A Q R Y 85 R Y Y A S L R H Y L N L V T R Q A R Y 86 R Y Y AS L R H Y L N L V T R Q R A Y 87 R Y Y A S L R H Y L N L V T R Q R Y A88 R Y Y A S L R H Y L N L V T R Q R Y 89 R Y Y A S L R H Y L N L V T RQ R Y 90 R Y Y A S L R H Y L N L V T R Q R Y 91 R Y Y A S L R H Y L N LV T R Q R Y 92 R Y Y A S L R H Y L N L V T R Q R Y 93 R Y Y A S L R H YL N L V T R Q R Y 94 R Y Y A S L R H Y L N L V T R Q R Y 95 R Y Y A S LR H Y L N L V T R Q R Y 96 R Y Y A S L R H Y L N L V T R Q R Y 97 R Y YA S L R H Y L N L V T R Q R Y 98 R Y Y A S L R H Y L N L V T R Q R Y 99R Y Y A S L R H Y L N L V T R Q R Y 100 R Y Y A S L R H Y L N L V T R QR Y 101 R Y Y A S L R H Y L N L V T R Q R Y 102 R Y Y A S L R H Y L N LV T R Q R Y 103 R Y Y A S L R H Y L N L V T R Q R Y 104 R Y Y A S L R HY L N L V T R Q R Y 105 R Y Y A S L R H Y L N L V T R Q R Y 106 R Y Y AS L R H Y L N L V T R Q R Y 107 A Y Y A S L R H Y L N L V T R Q R Y 108K Y Y A S L R H Y L N L V T R Q R Y 109 (Nme)A Y Y A S L R H Y L N L V TR Q R Y 110 R A Y A S L R H Y L N L V T R Q R Y 111 R Y A A S L R H Y LN L V T R Q R Y 112 R Y Y dA S L R H Y L N L V T R Q R Y 113 R Y Y A A LR H Y L N L V T R Q R Y 114 R Y Y A S A R H Y L N L V T R Q R Y 115 R YY A S L A H Y L N L V T R Q R Y 116 R Y Y A S L K H Y L N L V T R Q R Y117 R Y Y A S L R A Y L N L V T R Q R Y 118 R Y Y A S L R H A L N L V TR Q R Y 119 R Y Y A S L R H Y A N L V T R Q R Y 120 R Y Y A S L R H Y LA L V T R Q R Y 121 R Y Y A S L R H Y L N A V T R Q R Y 122 R Y Y A S LR H Y L N L A T R Q R Y 123 R Y Y A S L R H Y L N L V A R Q R Y 124 R YY A S L R H Y L N L V T A Q R Y 125 R Y Y A S L R H Y L N L V T K Q R Y126 R Y Y A S L R H Y L N L V T R A R Y 127 R Y Y A S L R H Y L N L V TR Q A Y 128 R Y Y A S L R H Y L N L V T R Q R A 129 R Y Y S A L R H Y IN L I T R Q R Y 130 R Y Y A S L R A Y L N L V T R Q R Y 131 R Y Y A S LR H Y L N L V T R Q R Y 132 R Y Y A S L R H Y L N L V T R Q R Y 133 R YY A S L R H Y L N L V T R Q R (Nme)Y 134 R Y Y A S L R H Y L N L V T R QR H 135 R Y Y A S L R A Y L N L V T R Q R H 136 R Y Y A S L R H Y L N LV T R Q R W 137 R Y Y A S L R H Y L N L V T R Q R F 138 R Y Y A S L R AY L N L V T R Q R F 139 R Y Y A S L R H Y L N L V T R Q R Y(CH2SO3) 140R Y Y A S L R A Y L N L V T R Q R P(OH) 141 R Y Y A S L R H Y L N L V TR Q hR Y 142 R Y Y A S L R H Y L N L V T R Q R Y 143 R Y Y A S L R A Y LN L V T R Q R Y 144 R Y Y A S L R A Y L N L V T R Q R Y 145 R Y Y A S LR H Y L N L V T R Q R Y 146 R Y Y A S L R H Y L N L V T R Q R Y 147 R YY A S(Ac) L R H Y L N L V T R Q R Y 148 hR Y Y A S L R H Y L N L V T R QR Y 149 R Y Y A S L hR H Y L N L V T R Q R Y 150 R Y Y A S L R H Y L N LV T R Q R Y 151 R Y Y A S L R H Y L N L V T R Q R Y 152 R Y Y A S L R HY L N L V T R Q R Y 153 R Y Y A S L R H Y L N L V T R Q R Y 154 R Y Y AS L R H Y L N L V T R Q R Y 155 R Y Y A S L R H Y L N L V T R Q R Y 156R Y Y A S L R H Y L N L V T R Q R Y 157 R Y Y A S L R H Y L N L V T R QR Y 158 R Y Y A S L R H Y L N L V T R Q R Y 159 R Y Y A S L R H Y L N LV T R Q R Y 160 R Y Y A S L R H Y L N L V T R Q R Y 161 hR Y Y A S L R HY L N L V T R Q R Y 162 hR Y Y A S L R H Y L N L V T R Q R Y 163 hR Y YA S L R H Y L N L V T R Q R Y 164 hR Y Y A S L R H Y L N L V T R Q R Y165 R Y Y A S L R H Y L N L V T R Q R Y 166 R Y Y A S L R A Y L N L V TR Q R Y 167 hR Y Y A S L R A Y L N L V T R Q R Y 168 hR Y Y A S L R A YL N L V T R Q R Y 169 R Y Y A S L R A Y L N L V T R Q R Y 170 R Y Y A SL K H Y L N L V T R Q R Y 171 R Y Y A S L R H Y L N L V T R Q R Y 172 RY Y A S L R A Y I N L I T R Q R Y 173 R Y Y A S L R H Y I N L I T R Q RY 174 R Y YY A S L R H Y L N A V T R Q R Y 175 R Y Y A S L R H Y L N L VT R Q R Y 176 R Y Y A S L R H Y L N L V T R Q R Y 177 R Y Y A S L R H YL N L V T R Q R Y 178 Y Y A S L R H Y L N L V T R Q R Y 179 R H Y L N LV T R Q R Y 180 R Y Y A S L R H Y L N L V T R Q R Y 181 R Y Y A S L R HY L N L V T R Q R Y 182 R Y Y A S L R H Y L N L V T R Q R Y 183Aminocaproyl A S L R H Y L N L V T R Q R Y 184 Aminocaproyl R H Y L N LV T R Q R Y 185 R Y Y A S L R H Y L N L V T R Q R Y 186 R Y Y A S L R HY L N L V T R Q R Y 187 R Y Y A S L R H Y L N L V T R Q R Y 188 R Y Y AS L R H Y L N L V T R Q R Y 189 K(PEG Y Y A S L R H Y L N L V T R Q R Y5000) 190 R Y Y A S L R H Y L N L V T R Q R Y 191 R Y Y A S L R H Y L NL V T R Q R Y 192 R Y Y A S(O- L R H Y L N L V T R Q R Y acyl- ationw/FA 193 K(Oct) Y Y A S L R H Y L N L V T R Q R Y 194 K(Oct) Y Y A S L RH Y L N L V T R Q R Y 195 R Y Y A S L R H Y L N L V T R Q R Y 196K(stea) Y Y A S L R H Y L N L V T R Q R Y 197 K(stea) Y Y A S L R H Y LN L V T R Q R Y 198 R Y Y A S L R H Y L N L V T R Q R Y 199 R Y Y A S LR H Y L N L V T R Q R Y 200 R Y Y A S L R H Y L N L V T R Q R Y 201 R YY A S L R H Y L N L V T R Q R Y 202 stearyl A S L R H Y L N L V T R Q RY 203 octyl A S L R H Y L N L V T R Q R Y 204 R Y Y A S L R H Y L N L VT R Q R Y 205 R Y Y A S L R H Y L N L V T R Q R Y 206 R Y Y A S L R H YL N L V T R Q R Y 207 R Y Y A S L R H Y L N L V T R Q R Y(8-Am-3,6-dioxaoct 208 R Y Y A S L R H Y L N L V T R Q R Y(11-Am- undecanoyl) 209R Y Y A S L R H Y L N L V T R Q R Y (12 Ado) 210 R Y Y A S L R H Y L N LV T R Q R Y (8-Oct) 211 + R Y Y A S L R H Y L N L V T R Q R Y 352 212 +R Y Y A S L R H Y L N L V T R Q R Y 353 213 + R Y Y A S L R H Y L N L VT R Q R Y 354 214 + R Y Y A S L R H Y L N L V T R Q R Y 355 215 + R Y YA S L R H Y L N L V T R Q R Y 356 216 + R Y Y A S L R H Y L N L V T R QR Y 357 217 + R Y Y A S L R H Y L N L V T R Q R Y 358 218 R Y Y A S L RH Y L N L V T R Q R Y 219 R Y Y A S L R H Y L N L V T R Q R Y 220 R Y YA S L R H Y L N L V T R Q R Y 221 R Y Y A S L R H Y L N L V T R Q R Y222 R Y Y A S L R H Y L N L V T R Q R Y 223 R Y Y A S L R H Y L N L V TR Q R Y 224 R Y Y A S L R H Y L N L V T R Q R Y 225 R Y Y A S L R H Y LN L V T R Q R Y 226 R Y Y A S L R H Y L N L V T R Q R Y 227 R Y Y A S LR H Y L N L V T R Q R Y 228 R Y Y A S L R H Y L N L V T R Q R Y 229 R YY A S L R H Y L N L V T R Q R Y 230 R Y Y A S L R H Y L N L V T R Q R Y231 + R Y Y A S L R H Y L N L V T R Q R Y 359 232 + R Y Y A S L R H Y LN L V T R Q R Y 360 233 + R Y Y A S L R H Y L N L V T R Q R Y 361 234 +R Y Y A S L R H Y L N L V T R Q R Y 362 235 + R Y Y A S L R H Y L N L VT R Q R Y 363 236 + R Y Y A S L R H Y L N L V T R Q R Y 364 237 + R Y YA S L R H Y L N L V T R Q R Y 365 238 R Y Y S A L R H Y I N L A T R Q RY 239 R Y Y S A L R H Y I N L I T R Q R Y 240 R Y Y S A L R H Y I N L IT R Q R Y 241 K Y Y S A L R H Y I N L I T R Q R Y 242 K Y Y S A L R H YI N L I T R Q R Y 243 R Y Y S A L R H Y I N L I T R Q R Y 244 R Y Y S AL R A Y I N L I T R Q R Y 245 R Y Y A S L R H Y I N L I T R Q R Y 246 RY Y A S L R H Y I N L I T R Q R Y 247 R Y Y S A L R A Y I N L I T R Q RY 248 R Y Y A S L R A Y I N L I T R Q R Y 249 R Y Y S A L R H Y I N L IT R Q R Y 250 R Y Y S A L R H Y I N L I T R Q R Y 251 R Y Y A S L R H YI N L I T R Q R Y 252 R Y Y A S L R H Y I N L I T R Q R Y 253 R Y Y A SL R H Y I N L I T R Q R Y 254 R Y Y A S L R H Y I N L I T R Q R Y 255 RY Y A S L R H Y I N L I T R Q R Y 256 R Y Y A S L R H Y I N L I T R Q RY 257 R Y Y A S L R H Y I N L I T R Q R Y 258 R Y Y S A L R A Y I N L IT R Q R Y 259 R Y Y S A L R A Y I N L I T R Q R Y 260 R Y Y A S L R A YI N L I T R Q R Y 261 R Y Y A S L R A Y I N L I T R Q R Y 262 R Y Y A SL R A Y I N L I T R Q R Y 263 R Y Y A S L R A Y I N L I T R Q R Y 264 RY Y A S L R A Y I N L I T R Q R Y 265 R Y Y A S L R A Y I N L I T R Q RY 266 R Y Y A S L R A Y I N L I T R Q R Y 267 R Y Y A S L R H Y I N L IT R Q R Y 268 R Y Y A S L R A Y I N L I T R Q R Y 269 R Y Y A S L R H YI N L I T R Q R Y 270 R Y Y A S L R H Y I N L I T R Q R Y 271 R Y Y A SL R H Y I N L I T R Q R Y 272 R Y Y S A L R H Y I N L I T R Q R Y 273 RY Y A S L R A Y I N L I T R Q R Y 274 R Y Y A S L R A Y I N L I T R Q RY 275 R Y Y A S L R A Y I N L I T R Q R Y 276 R Y Y A S L R A Y I N L IT R Q R Y 277 R Y Y A S L R A Y I N L I T R Q R Y 278 R Y Y A S L R H YI N L I T R Q R Y 279 R Y Y A S L R A Y I N L I T R Q R Y 280 R Y Y A SL R H Y I N L I T R Q R Y 281 R Y Y S A L R H Y I N L I T R Q R Y 282 RY Y S A L R H Y I N L I T R Q R Y 283 R Y Y S A L R H Y I N L I T R Q RY 284 R Y Y S A L R H Y I N L I T R Q R Y 285 R Y Y A S L R H Y L N L VT R Q R Y 286 R Y Y A S L R H Y L N L V T R Q R Y 287 R Y Y A S L R H YL N L V T R Q R Y 288 R Y Y A S L R H F L N L V T R Q R Y 289 R Y Y A SL R H Y L N L A P R Q R Y 290 R Y Y A S L R H Y L N L A Aib R Q R Y 291R Y Y A S L R H Y L N L A Sar R Q R Y 292 R Y Y S A L R H Y L N L V T RQ R Y 293 K Y Y A S L R H Y L N L V T R Q R Y 294 R Y Y A S L R H Y L NL V T R Q R Y 295 R Y Y A S L R H Y L N L V T R Q R Y 296 R Y Y A S L RH Y L N L V T R Q R Y 297 R Y Y A S L R H Y L N L V T R Q R Y 298 R Y YA S L R H Y L N L V T R Q R Y 299 R Y Y A S L R H Y L N L V T R Q R Y300 R Y Y A S L R H Y L N L V T R Q R Y 301 R Y Y A S L R H Y L N L V TR Q R Y 302 R Y Y A S L R H Y L N L V T R Q R Y 303 R Y Y A S L R H Y LN L V T R Q R Y 304 hR Y Y A S L R H Y L N L V T R Q R Y 305 hR Y Y A SL R H Y L N L V T R Q R Y 306 hK Y Y A S L R H Y L N L V T R Q R Y 307 RY Y A S L R H Y L N L V T R Q R Y 308 R Y Y A S L R A Y L N L V T R Q RY 309 Q Y A A D L R R Y I N M L T R P R Y 310 Q Y A A D L R R Y I N M LT R P R Y 311 Q Y A A D L R R Y I N M L T R Q R Y 312 Q Y A A D L R R YI N M L T R Q R Y 313 Q Y A A S L R H Y I N M L T R Q R Y 314 Q Y A S AL R H Y I N M L T R Q R Y 315 Q Y A A D L R H Y I N M L T R Q R Y 316 QY A A D L R R Y I N M L T R Q R Y 317 Q Y A A S L R R Y I N M L T R Q RY 318 Q Y A A D L R R Y I N M L T R Q R Y 319 Q Y A A D L R R Y I N M LT R Q R Y 320 Q Y A A D L R R Y I N M L T R Q R Y 321 Q Y A A D L hK R YI N M L T R Q R Y 322 Q Y A A D L hR R Y I N M L T R Q R Y 323 Q Y A A DL Om R Y I N M L T R Q R Y 324 Q Y A A D L Ot R Y I N M L T R Q R Y 325Q Y A A D L R hK Y I N M L T R Q R Y 326 Q Y A A D L R hR Y I N M L T RQ R Y 327 Q Y A A D L R Om Y I N M L T R Q R Y 328 Q Y A A D L R Ot Y IN M L T R Q R Y 329 Q Y A A D L hR hR Y I N M L T R Q R Y 330 Q Y A A DL R R Y I N M L T R Q R Y 331 Q Y A A D L R R Y I N M L T R Q R Y 332 QY A A D L R R Y I N M L T R Q R Y 333 Q Y A A D L R R Y I N M L T R Q RY 334 Q Y A A D L R R Y I N M L T R Q R Y 335 Q Y A A D L R R Y I N M LT R Q R Y 336 Q Y A A D L R R Y I N M L T R Q R Y 337 Q Y A A D L R R YI N M L T R Q R Y 338 Q Y A A D L R R Y I N M L T R Q R Y 339 Q Y A A DL R R Y I N M L T R Q R Y 340 Q Y A A D L R R Y I N M L T R Q R Y 341 QY A A D L R R Y I N M L T R Q R Y 342 Q Y A A D L R R Y I N M L T R Q RY 343 Q Y A A D L R R Y I N M L T R Q R Y 344 Q Y A A D L R R Y I N M LT R Q R Y 345 Q Y A A D L R R Y I N M L T R Q R Y 346 Q Y A A D L R R YI N M L T R Q R Y 347 Q Y A A E L R R Y I Q M L T R Q R Y 348 349 350351 T R Q R 436 R Y Y A S L R H Y L N L V T R Q R Y 437 R Y Y A S L R AY I N L I T R Q R Y 438 R Y Y A S L R A Y I N L I T R Q R Y 439 R Y Y AS L R A Y I N L I T R Q R Y 440 K Y Y A S L R A Y I N L I T R Q R Y 441K Y Y A S L R A Y I N L I T R Q R Y 442 K Y Y A S L R A Y I N L I T R QR Y 443 R Y Y A S L R H Y L N L V T R Q R Y 444 R Y Y A S L R H Y L N LV T R Q R Y 445 R Y Y A S L R A Y I N L V T R Q R Y 446 R Y F A S L R AY I N L V T R Q R Y 447 R Y K A S L R A Y L N L V T R Q R Y 448 R Y F AS L R A Y I N L V T R Q R Y 449 R Y K A S L R A Y L N L V T R Q R Y 450R Y Y A S L R A Y L N L V T R Q R W 451 A(Nme) Y Y A S L R H Y L N L V TR Q R Y 452 R Y Y A S L R H Y L N L V T R Q R Y 453 R Y Y A S L R H Y LN L V T R Q R Y 454 R Y K A S L R A Y I N L I T R Q R Y 455 R Y Y A S LR A Y I N L I T R Q R Y 456 R Y Y A S L R A Y I N L I T R Q R Y 457 R YY A S L R A Y I N L I T R Q R Y 458 R Y Y A S L R A Y I N L I T R Q R F459 R Y Y A S L R A Y I N L I T R Q R W 460 R Y Y A S L R A Y I N L I TR Q R Y 461 R Y Y A S L R A Y I N L I T R Q R Y 462 R Y Y A S L R A Y IN L I T R Q R Y 463 R Y Y A S L R A Y I N L I T R Q R Y 464 R Y Y A S LR A Y I N L I T R Q R Y 465 R Y Y A S L R A Y I N L I T R Q R Y 466 R YY A S L R A Y I N L I T R Q R Y 467 R Y Y A S L R A Y I N L I T R Q R Y468 R Y Y A S L R A Y I N L I T R Q R Y 469 R Y Y A S L R A Y I N L I TR Q R Y 470 K Y Y A S L R A Y I N L I Y R Q R Y 471 K Y Y A S L R A Y IN L I T R Q R Y 472 K Y Y A S L R A Y I N L I T R Q R Y 473 R Y Y A S LR A Y I N L I Y R Q R Y 474 R Y Y A S L R A Y I N L I T R Q R Y 475 Q VA A D L R R Y I N M L T R Q R Y 476 Q Y A A D L R R Y I N M L Y R Q R Y477 Q Y A A D L R R Y I N M L T R Q R Y 478 Q V A A D L R R Y I N M L TR Q R Y 479 R V V A S L R A Y I N L I T R Q R Y 480 R Y Y A S L R A Y IN L I Y R Q R Y 481 482

While the present invention has been described in terms of severalexamples and embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationswhich come within the scope of the invention as claimed.

What is claimed is:
 1. A method for treating Type-2 diabetes, obesity,or insulin-resistance syndrome, or for causing weight loss, in a mammalin need thereof, the method comprising administering to the mammal atherapeutically effective amount of a PPF polypeptide to treat obesity,Type-2 diabetes, or insulin-resistance syndrome, or to cause weightloss, wherein the PPF polypeptide comprises the amino acid sequence ofFormula (VII) (SEQ ID NO: 482):Xaa₁ Xaa₂ Pro Xaa₄ Pro Xaa₆ His Pro Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ AlaXaa₁₉ Tyr Xaa₂₁ Xaa₂₂ Xaa₂₃ Leu Xaa₂₅ Xaa₂₆ Xaa₂₇Xaa₂₈ Xaa₂₉ Xaa₃₀ Xaa₃₁ Thr Arg Gln Arg Tyr

wherein: Xaa₁ is absent; Xaa₂ is absent; Xaa₄ is Lys; Xaa₆ is Glu; Xaa₉is Gly; Xaa₁₀ is Glu; Xaa₁₁ is Asp; Xaa₁₂ is Ala; Xaa₁₃ is Ser or Pro;Xaa₁₄ is Pro, Ala, or Ser; Xaa₁₅ is Glu; Xaa₁₆ is Glu; Xaa₁₇ is Leu;Xaa₁₉ is Arg, Lys; Xaa₂₁ is Tyr; Xaa₂₂ is Ala; Xaa₂₃ is Ser; Xaa₂₅ isArg; Xaa₂₆ is Ala; Xaa₂₇ is Tyr; Xaa₂₈ is Ile; Xaa₂₉ is Asn; Xaa₃₀ isLeu; and Xaa₃₁ is Ile.
 2. The method of claim 1 for treating Type 2diabetes.
 3. The method of claim 1 for treating obesity in the mammal inneed thereof.
 4. The method of claim 1, wherein the PPF polypeptidecomprises the amino acid sequence set forth in SEQ ID NO:
 438. 5. Themethod of claim 1, wherein the PPF polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 266, 437,438, 439, 442, 462, 469, 470, 471, and
 472. 6. The method of claim 1,wherein the PPF polypeptide further comprises an N-terminal cap.
 7. Themethod of claim 1, wherein the PPF polypeptide is linked to one or morewater-soluble polymers.
 8. The method of claim 7, wherein the polymer isselected from at least one of the group consisting of polyethyleneglycol and a fatty acid molecule, wherein the polymer is linked to theN- or C-terminus of the polypeptide, or the side chain of a lysine orserine amino acid residue within the sequence of the polypeptide.